Compounds for inhibiting NS3 and compositions containing the inhibited protein

ABSTRACT

Imidazole-based compounds as hepatitis C virus (HCV) inhibitors. The compounds have an imidazole core that is disubstituted via amide links. Also described are a pharmaceutical composition incorporating the imidazole-based compound, a method of preparing these compounds, and a method for using the pharmaceutical composition in the treatment of HCV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of Ser. No. 16/384,472, havinga filing date of Apr. 15, 2019.

STATEMENT OF FUNDING ACKNOWLEDGEMENT

This project was funded by the National Plan for Science, Technology andInnovation (MAARIFAH), King Abdulaziz City for Science and Technology,the Kingdom of Saudi Arabia under award number 12-BIO3193-03. Theproject was also supported by the Science and Technology Unit of KingAbdulaziz University.

BACKGROUND OF THE INVENTION Technical Field

The present disclosure relates to a family of disubstituted imidazolecompounds. A pharmaceutical composition involving the compounds, amethod of preparing the compounds, and a method of treating orinhibiting hepatitis C infection via inhibiting the functioning of theNS3 protease using the compounds are disclosed.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

Hepatitis C is a life-threatening viral infection that is wide spread inthe world [Organization, W. H., Global hepatitis report 2017. 2017].Initially, the virus infects liver cells but remains asymptomatic for anextended period [Dickson, R. C., Clinical manifestations of hepatitis C.Clinics in liver disease 1997, 1 (3), 569-85]. This is anepidemiological challenge because patients diagnosed with hepatitis Ccome from two pools; recently-infected and later-stage asymptomatic. Theinitial symptoms of hepatitis C such as fever, fatigue, nausea, andliver tenderness can be misleading because they are tolerated by mostpatients. After several years of infection, the virus activates,replicates, and causes complications that start with liver scarring,fibrosis followed by cirrhosis and eventually liver failure andcarcinoma [Friedrich, M. J., Third millennium challenge: hepatitis C.Jama 1999, 282 (3), 221-2]. The hepatitis C virus (HCV) is usuallytransmitted via the blood of an infected person [Bocket, L.; Chevaliez,S.; Talbodec, N.; Sobaszek, A.; Pawlotsky, J. M.; Yazdanpanah, Y.,Occupational transmission of hepatitis C virus resulting from use of thesame supermarket meat slicer. Clinical microbiology and infection: theofficial publication of the European Society of Clinical Microbiologyand Infectious Diseases 2011, 17 (2), 238-41]. Awareness of hepatitis Chealth problems and forceful efforts to combat the spread on HCV inhealthcare settings have led to a significant decline in new infectioncases [Organization, W. H., Global hepatitis report 2017. 2017; andHatzakis, A. et al. The present and future disease burden of hepatitis Cvirus (HCV) infections with today's treatment paradigm—volume 2. J ViralHepat 2015, 22 Suppl 1, 26-45]. However, the death toll from hepatitis Ccomplications remains as high as 400,000 annually due to the abovedescribed unique clinical vs. epidemic profile of hepatitis C. Anestimated 70 million people globally are infected with HCV, constitutinga major health problem [Collaborators, P. O. H., Global prevalence andgenotype distribution of hepatitis C virus infection in 2015: amodelling study. Lancet Gastroenterol Hepatol 2017, 2 (3), 161-176]. In2013, the death rate from hepatitis C complications surpassed that ofHIV. In 2015, hepatitis C-related deaths exceeded those of tuberculosisand malaria combined [Stanaway, J. D. et al. The global burden of viralhepatitis from 1990 to 2013: findings from the Global Burden of DiseaseStudy 2013. Lancet (London, England) 2016, 388 (10049), 1081-1088].

Up until 2013, interferon (or pegylated interferon) and ribavirin werethe most effective available therapies, however the long-term efficacieswere as low as 3% [North, C. S.; Hong, B. A.; Adewuyi, S. A.; Pollio, D.E.; Jain, M. K.; Devereaux, R.; Quartey, N. A.; Ashitey, S.; Lee, W. M.;Lisker-Melman, M., Hepatitis C treatment and SVR: the gap betweenclinical trials and real-world treatment aspirations. General hospitalpsychiatry 2013, 35 (2), 122-8]. Identification of the hepatitis Cgenome and proteome in early 1990s prompted research efforts that led todevelopment of direct antiviral agents (DAA) in 2011 includingbocepreivir and telaprevir. The DAAs revolutionized the treatment of HCVinfection⁹⁻¹¹ and the current situation of the treatment lookspromising. Nonetheless, new therapies will likely be needed to addressemerging resistance of this rapidly mutating virus [Bartenschlager, R.;Baumert, T. F.; Bukh, J.; Houghton, M.; Lemon, S. M.; Lindenbach, B. D.;Lohmann, V.; Moradpour, D.; Pietschmann, T.; Rice, C. M.; Thimme, R.;Wakita, T., Critical challenges and emerging opportunities in hepatitisC virus research in an era of potent antiviral therapy: Considerationsfor scientists and funding agencies. Virus research 2018, 248, 53-62].

The HCV genome is a positive-sense, single-stranded RNA virus belongingto flaviviridae [Murray, C. L.; Jones, C. T.; Rice, C. M., Architects ofassembly: roles of Flaviviridae non-structural proteins in virionmorphogenesis. Nature reviews microbiology 2008, 6 (9), 699-708]. It iscomposed of about 9,400 nucleotides with highly conserved 5′ and 3′terminal regions. The untranslated 5′ terminus is followed by a singleopen reading frame that encodes a polyprotein of 3010 to 3303 aminoacids. The virus genome translates mainly to structural andnon-structural proteins. The non-structural (NS) proteins including NS3,NS4A, NS4B, NS5A and NS5B comprise proteins that are important formaturation and replication of the virus.

NS3 dually functions as protease (N-terminal domain) and RNA helicase(C-terminal domain). When conformed as protease, NS3 catalyzes theprocessing of the viral proteome to functional proteins by cleavingNS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions. NS5A is anon-enzymatic protein that is important for viral replication howeverthe exact mechanism is poorly understood. NS5B is well characterized tobe the viral RNA polymerase. NS4A is a small (54 amino acids) andversatile multi-functioning peptide. It acts as the activating cofactorof NS3 protease (N-terminal) and NS3 helicase (C-terminal) [Ishido, S.;Fujita, T.; Hotta, H., Complex formation of NS5B with NS3 and NS4Aproteins of hepatitis C virus. Biochemical and biophysical researchcommunications 1998, 244 (1), 35-40; and Kim, D. W.; Gwack, Y.; Han, J.H.; Choe, J., C-terminal domain of the hepatitis C virus NS3 proteincontains an RNA helicase activity. Biochemical and biophysical researchcommunications 1995, 215 (1), 160-166]. It is also important for theintegration of NS3 to the host cell endoplasmic reticulum [Wölk, B.;Sansonno, D.; Krausslich, H.-G.; Dammacco, F.; Rice, C. M.; Blum, H. E.;Moradpour, D., Subcellular localization, stability, and trans-cleavagecompetence of the hepatitis C virus NS3-NS4A complex expressed intetracycline-regulated cell lines. Journal of virology 2000, 74 (5),2293-2304] and neutralization of the immune response of the host celltowards the viral invasion [Li, K.; Foy, E.; Ferreon, J. C.; Nakamura,M.; Ferreon, A. C.; Ikeda, M.; Ray, S. C.; Gale, M.; Lemon, S. M.,Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage ofthe Toll-like receptor 3 adaptor protein TRIF. Proceedings of theNational Academy of Sciences 2005, 102 (8), 2992-2997; and Meylan, E.;Curran, J.; Hofmann, K.; Moradpour, D.; Binder, M.; Bartenschlager, R.;Tschopp, J., Cardif is an adaptor protein in the RIG-I antiviral pathwayand is targeted by hepatitis C virus. Nature 2005, 437 (7062), 1167].The current approved DAAs elevated the patient sustained cure to 95% inless than two months of use [Kardashian, A. A.; Pockros, P. J., Novelemerging treatments for hepatitis C infection: a fast-moving pipeline.Therapeutic advances in gastroenterology 2017, 10 (2), 277-282]. Drugsavailable for prescription are categorized to three classes: NS3/4Ainhibitors, e.g. telaprevir, boceprovir, simeprevir, asunaprevir,paritaprevir, grazopreivir, and voxilaprevir (binding at the substratesite), NS5A inhibitors, e.g. daclatasvir, ledipasvir, ombitasvir,elbasvir, and velpatasvir, and NS5B inhibitors, e.g. soforbuvir, anddasabuvir [Feld, J. J., Direct-Acting Antivirals for Hepatitis C Virus(HCV): The Progress Continues. Current drug targets 2017, 18 (7),851-862].

The use of DAA combinations with high rates of sustained viral recovery(SVR) became routine in therapy regimens [Aghemo, A.; Piroth, L.;Bhagani, S., What do clinicians need to watch for with direct-actingantiviral therapy? Journal of the International AIDS Society 2018, 21Suppl 2, e25076]. For instance, a combination of voxilaprevir (NS3/4Ainhibitor), velpatasvir (NS5A inhibitor), and sofosbuvir (NS5Binhibitor) that covers all HCV genotypes 1-6 has been prescribed topatients suffering relapse subsequent to failure of DAA monotherapy[Chahine, E. B.; Kelley, D.; Childs-Kean, L. M.,Sofosbuvir/Velpatasvir/Voxilaprevir: A Pan-Genotypic Direct-ActingAntiviral Combination for Hepatitis C. The Annals of pharmacotherapy2018, 52 (4), 352-363].

As outlined in the World Health Organization Global Hepatitis Report,there are several remaining challenges that must be met to eradicate HCVby 2030 [Organization, W. H., Global hepatitis report 2017. 2017]: 1)access to the treatment in economically-challenged areas must beincreased [Chhatwal, J.; Chen, Q.; Aggarwal, R., Estimation of HepatitisC Disease Burden and Budget Impact of Treatment Using Health EconomicModeling. Infect Dis Clin North Am 2018, 32 (2), 461-480]; 2) emergingdrug resistance must be addressed [Cuypers, L.; Libin, P.; Schrooten,Y.; Theys, K.; Di Maio, V. C.; Cento, V.; Lunar, M. M.; Nevens, F.;Poljak, M.; Ceccherini-Silberstein, F., Exploring resistance pathwaysfor first-generation NS3/4A protease inhibitors boceprevir andtelaprevir using Bayesian network learning. Infection, Genetics andEvolution 2017, 53, 15-23]; 3) efficacy in broader sectors of HCVpatients, such as elder and impaired kidney or liver patients, must beincreased [Hellard, M. E.; Chou, R.; Easterbrook, P., WHO guidelines ontesting for hepatitis B and C-meeting targets for testing. BioMedCentral: 2017]. Thus, there is a clear need to identify new approachesto inhibit viral protein targets and treat HCV infection. The currentdisclosure introduces a new class of compounds that interfere with NS4Ain its binding with NS3 protease and deactivate the enzyme [Joyce, M.;Williams, M.; Hindsgaul, O.; Tyrrell, D. Inhibitors of hepatitis C virusprotease, U.S. patent application Ser. No. 10/319,402].

In view of the forgoing, one objective of the present disclosure is toprovide therapeutic di-substituted imidazole-based compounds and aprocess of synthesizing these compounds. A further objective of thepresent disclosure is to provide a pharmaceutical composition comprisingthe imidazole-based compound and a method of treating or preventing HCVinfection.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, the present disclosure relates to acompound of formula (I)

or a salt thereof, a solvate thereof, a tautomer thereof, a stereoisomerthereof, or a mixture thereof, wherein (i) R₁ is selected from the groupconsisting of an optionally substituted alkyl, an optionally substitutedcycloalkyl, an optionally substituted arylalkyl, and an optionallysubstituted aryl, and (ii) m is an integer in a range of 2-8.

In one embodiment, R₁ is an optionally substituted C₄₋₁₀ alkyl.

In one embodiment, R₁ is a C₄-C₁₀ alkyl optionally substituted with atleast one substituent selected from the group consisting of an alkyloxy,a cycloalkyloxy, an aryloxy, an amine, and an amide.

In one embodiment, R₁ is a linear C₄₋₁₀ alkyl.

In one embodiment, R₁ is at least one selected from the group consistingof n-pentyl, n-hexyl, n-heptyl, 2-(cyclohexyloxy)ethyl,3-(cyclohexyloxy)propyl, 2-phenoxyethyl, 3-phenoxypropyl,4-isopropoxybutyl, 6-methylheptyl, and 3-methylpentyl.

In one embodiment, R₁ is n-pentyl.

In one embodiment, R₁ is n-hexyl.

In one embodiment, m is 4.

In one embodiment, the compound of formula (I) is

According to a second aspect, the present disclosure relates to acompound of formula (II)

or a salt thereof, a solvate thereof, a tautomer thereof, a stereoisomerthereof, or a mixture thereof, wherein (i) R₂ is selected from the groupconsisting of an optionally substituted amide, an optionally substitutedaryl, and an optionally substituted heteroaryl, R₃ is selected from thegroup consisting of an optionally substituted alkyl, an optionallysubstituted cycloalkyl, an optionally substituted arylalkyl, anoptionally substituted alkenyl, and an optionally substituted aryl, and(iii) n is an integer in a range of 1-4.

In one embodiment, R₂ is selected from the group consisting of anunsubstituted amide (—C(O)NH₂), N-methylamide (—C(O)NHCH₃), a pyridyl,and a furyl.

In one embodiment, R₃ is selected from the group consisting of n-pentyl,n-hexyl, n-heptyl, 2-(cyclohexyloxy)ethyl, 3-(cyclohexyloxy)propyl,2-phenoxyethyl, 3-phenoxypropyl, 4-isopropoxybutyl, 6-methylheptyl,3-methylpentyl, 2-acetamidoethyl, and 3,7-dimethylocta-2,6-dien-1-yl.

According to a third aspect, the present disclosure relates to apharmaceutical composition involving the compound of formula (I) of thefirst aspect and a pharmaceutically acceptable carrier and/or excipient.

In one embodiment, the pharmaceutical composition contains 0.5-500 M ofthe compound of formula (I) relative to a total volume of thecomposition.

In one embodiment, the pharmaceutically acceptable carrier and/orexcipient is at least one selected from the group consisting of abuffer, an inorganic salt, a fatty acid, a vegetable oil, a syntheticfatty ester, a surfactant, and a polymer.

In one embodiment, the pharmaceutical composition further includes anantiviral agent.

In one embodiment, the compound of formula (I) is

According to a fourth aspect, the present disclosure relates to a methodof preventing or treating hepatitis C virus (HCV) infection. The methodinvolves administering the pharmaceutical composition of the thirdaspect to a subject in need of therapy.

In one embodiment, 0.1-100 mg/kg of the compound of formula (I) isadministered per body weight of the subject.

In one embodiment, the compound of formula (I) binds to an activationsite of hepatitis C virus (HCV) serine protease.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a schematic diagram illustrating the design of imidazolescaffolds based on the planar region of NS4A.

FIG. 2 is a synthesis scheme for the preparation of starting materialsand compounds of formulae (I) and (II).

FIG. 3 shows a list of MOC compounds of the present disclosure.

FIG. 4 shows the purity of NS3 using SDS-Page electrophoresis method.

FIG. 5 shows a competition assay using fluorescence anisotropy thatdetermines ability of compounds of formula (I) replacing FITC-NS4A inbinding with NS3.

FIG. 6 is an enzyme inhibition assay that shows the effect of compoundMOC-23 on NS3 activity.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the disclosure are shown.

As used herein, the words “a” and “an” and the like carry the meaning of“one or more”. Within the description of this disclosure, where anumerical limit or range is stated, the endpoints are included unlessstated otherwise. Also, all values and subranges within a numericallimit or range are specifically included as if explicitly written out.

As used herein, the terms “complex”, “compound”, and “product” are usedinterchangeably, and are intended to refer to a chemical entity, whetherin the solid, liquid or gaseous phase, and whether in a crude mixture orpurified and isolated.

As used herein, the term “solvate” refers to a physical association of acompound of this disclosure with one or more solvent molecules, whetherorganic or inorganic. This physical association includes hydrogenbonding. In certain instances, the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. The solvent molecules in thesolvate may be present in a regular arrangement and/or a non-orderedarrangement. The solvate may comprise either a stoichiometric ornonstoichiometric amount of the solvent molecules. Solvate encompassesboth solution phase and isolable solvates. Exemplary solvents include,but are not limited to, water, methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, tert-butanol, ethyl acetate andother lower alkanols, glycerine, acetone, dichloromethane (DCM),dimethyl sulfoxide (DMSO), dimethyl acetate (DMA), dimethylformamide(DMF), isopropyl ether, acetonitrile, toluene, N-methylpyrrolidone(NMP), tetrahydrofuran (THF), tetrahydropyran, other cyclic mono-, di-and tri-ethers, polyalkylene glycols (e.g. polyethylene glycol,polypropylene glycol, propylene glycol), and mixtures thereof insuitable proportions. Exemplary solvates include, but are not limitedto, hydrates, ethanolates, methanolates, isopropanolates and mixturesthereof. Methods of solvation are generally known to those of ordinaryskill in the art.

As used herein, the term “tautomer” refers to constitutional isomers oforganic compounds that readily convert by tautomerization ortautomerism. The interconversion commonly results in the formalmigration of a hydrogen atom or proton, accompanied by a switch of asingle bond and adjacent double bond. Tautomerism is a special case ofstructural isomerism, and because of the rapid interconversion,tautomers are generally considered to be the same chemical compound. Insolutions in which tautomerization is possible, a chemical equilibriumof the tautomers will be reached. The exact ratio of the tautomersdepends on several factors including, but not limited to, temperature,solvent and pH. Exemplary common tautomeric pairs include, but are notlimited to, ketone and enol, enamine and imine, ketene and ynol, nitrosoand oxime, amide and imidic acid, lactam and lactim (an amide and imidictautomerism in heterocyclic rings), and open-chain and cyclic forms ofan acetal or hemiacetal (e.g., in reducing sugars).

As used herein, the term “stereoisomer” refers to isomeric moleculesthat have the same molecular formula and sequence of bonded atoms (i.e.constitution), but differ in the three-dimensional orientations of theiratoms in space. This contrasts with structural isomers, which share thesame molecular formula, but the bond connection of their order differs.By definition, molecules that are stereoisomers of each other representthe same structural isomer. Enantiomers are two stereoisomers that arerelated to each other by reflection, they are non-superimposable mirrorimages. Every stereogenic center in one has the opposite configurationin the other. Two compounds that are enantiomers of each other have thesame physical properties, except for the direction in which they rotatepolarized light and how they interact with different optical isomers ofother compounds. Diastereomers are stereoisomers not related through areflection operation, they are not mirror images of each other. Theseinclude meso compounds, cis- and trans- (E- and Z-) isomers, andnon-enantiomeric optical isomers. Diastereomers seldom have the samephysical properties. In terms of the present disclosure, stereoisomersmay refer to enantiomers, diastereomers, or both.

Conformers, rotamers, or conformational isomerism refers to a form ofisomerism that describes the phenomenon of molecules with the samestructural formula but with different shapes due to rotations around oneor more bonds. Different conformations can have different energies, canusually interconvert, and are very rarely isolatable. There are somemolecules that can be isolated in several conformations. Atropisomersare stereoisomers resulting from hindered rotation about single bondswhere the steric strain barrier to rotation is high enough to allow forthe isolation of the conformers. In terms of the present disclosure,stereoisomers may refer to conformers, atropisomers, or both.

In terms of the present disclosure, stereoisomers of the double bonds,ring systems, stereogenic centers, and the like can all be present inthe compounds, and all such stable isomers are contemplated in thepresent disclosure. Cis- and trans- (or E- and Z-) stereoisomers of thecompounds of the present disclosure wherein rotation around the doublebond is restricted, keeping the substituents fixed relative to eachother, are described and may be isolated as a mixture of isomers or asseparated isomeric forms. S- and R- (or L- and D-) stereoisomers of thecompounds of the present disclosure are described and may be isolated asa mixture of isomers or as separated isomeric forms. All processes ormethods used to prepare compounds of the present disclosure andintermediates made therein are considered to be part of the presentdisclosure. When stereoisomeric products are prepared, they may beseparated by conventional methods, for example, by chromatography,fractional crystallization, or use of a chiral agent.

As used herein, the term “substituted” refers to at least one hydrogenatom that is replaced with a non-hydrogen group, provided that normalvalencies are maintained and that the substitution results in a stablecompound. When a substituent is noted as “optionally substituted”, thesubstituents are selected from halo, hydroxyl, alkoxy, oxo, alkanoyl,aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino,disubstituted amines (e.g. in which the two amino substituents areselected from the exemplary group including, but not limited to, alkyl,aryl or arylalkyl), alkanoylamino, aroylamino, aralkanoylamino,substituted alkanoylamino, substituted arylamino, substitutedaralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, alkylthiono,arylthiono, aryalkylthiono, alkylsulfonyl, arylsulfonyl,arylalkylsulfonyl, sulfonamide (e.g. —SO₂NH₂), substituted sulfonamide,nitro, cyano, carboxy, unsubstituted amide (i.e. —CONH₂), substitutedamide (e.g. —CONHalkyl, —CONHaryl, —CONHarylalkyl or cases where thereare two substituents on one nitrogen from alkyl, aryl, or alkylalkyl),alkoxycarbonyl, aryl, substituted aryl, guanidine, heterocyclyl (e.g.indolyl, imidazoyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl,pyrimidiyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl,homopiperazinyl and the like), substituted heterocyclyl and mixturesthereof. The substituents may themselves be optionally substituted, andmay be either unprotected, or protected as necessary, as known to thoseskilled in the art, for example, as taught in Greene, et al.,“Protective Groups in Organic Synthesis”, John Wiley and Sons, SecondEdition, 1991, hereby incorporated by reference in its entirety.

As used herein, the term “alkyl” unless otherwise specified refers toboth branched and straight chain saturated aliphatic primary, secondary,and/or tertiary hydrocarbons of typically C₁ to C₂₁, for example C₁, C₂,C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, and specificallyincludes, but is not limited to, methyl, trifluoromethyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl,isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl,3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylhexyl,heptyl, octyl, nonyl, 3,7-dimethyloctyl, decyl, undecyl, dodecyl,tridecyl, 2-propylheptyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,octadecyl, nonadecyl, and eicosyl.

The term “cycloalkyl” refers to cyclized alkyl groups. Exemplarycycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and adamantyl. Branchedcycloalkyl groups such as exemplary 1-methylcyclopropyl and2-methylcyclopropyl groups are included in the definition of cycloalkylas used in the present disclosure.

The term “alkenyl” refers to a straight, branched, or cyclic hydrocarbonfragment containing at least one C═C double bond. Exemplary alkenylgroups include, without limitation, 1-propenyl, 2-propenyl (or “allyl”),1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl,1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl,1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl,7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl,6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl,4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, and 9-decenyl.

The term “arylalkyl”, as used herein, refers to a straight or branchedchain alkyl moiety having 1 to 8 carbon atoms that is substituted by anaryl group as defined herein, and includes, but is not limited to,benzyl, phenethyl, 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl,2,4-dimethylbenzyl, 2-(4-ethylphenyl)ethyl, 3-(3-propylphenyl)propyl,and the like.

The term “aryl”, as used herein, and unless otherwise specified, refersto phenyl, biphenyl, naphthyl, anthracenyl, and the like.

The term “heteroaryl” refers to an aryl group where at least one carbonatom is replaced with a heteroatom (e.g. nitrogen, oxygen, sulfur) andcan be indolyl, furyl, imidazolyl, triazolyl, triazinyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrrolyl, pyrazinyl,tetrazolyl, pyridyl (or its N-oxide), thienyl, pyrimidinyl (or itsN-oxide), 1H-indolyl, isoquinolyl (or its N-oxide), or quinolyl (or itsN-oxide), for example.

The terms “alkoxy” and “alkyloxy” refer to a straight or branched alkylgroup attached to an oxygen atom. Exemplary alkyloxy groups include, butare not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, secondary butoxy, tertiary butoxy, pentoxy, isopentoxy,hexyloxy, heptyloxy, octyloxy, nonyloxy, and decyloxy.

The terms “cycloalkoxy” and ‘cycloalkyloxy” as used herein means acycloalkyl group linked to an oxygen atom, such as, for example,cyclobutyloxy or cyclopropyloxy.

The term “aryloxy” refers to an aryl group bonded to an oxygen atom.Exemplary aryloxy groups include, but are not limited to, phenoxy,4-methylphenoxy, and naphthaloxy.

The term “amide”, as used herein, and unless otherwise specified, refersto an amide (—C(O)NR_(c)R_(d)) that is unsubstituted (—C(O)NH₂),monosubstituted (where R is a hydrogen), or disubstituted where R_(c)and R_(d) are independently an optionally substituted alkyl, anoptionally substituted cycloalkyl, an optionally substituted arylalkyl,or an optionally substituted aryl.

As used herein, the term “amine” includes unsubstituted amine (—NH₂),monosubstituted amine (—NHR_(a)), as well as disubstituted amine(—NR_(a)R_(b)), wherein R_(a) and R_(b) are independently an optionallysubstituted alkyl, an optionally substituted cycloalkyl, an optionallysubstituted arylalkyl, or an optionally substituted aryl.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample, and without limitation, isotopes of hydrogen include deuteriumand tritium, isotopes of carbon include ¹³C and ¹⁴C, isotopes ofnitrogen include ¹⁴N and ¹⁵N, and isotopes of oxygen include ¹⁶O, ¹⁷Oand ¹⁸O. Isotopically labeled compounds of the disclosure can generallybe prepared by conventional techniques known to those skilled in the artor by processes and methods analogous to those described herein, usingan appropriate isotopically labeled reagent in place of the non-labeledreagent otherwise employed.

According to a first aspect, the present disclosure relates to acompound of formula (I)

or a salt thereof, a solvate thereof, a tautomer thereof, a stereoisomerthereof, or a mixture thereof.

R₁ is selected from the group consisting of an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, and an optionally substituted aryl.

In one or more embodiments, R₁ is an optionally substituted alkyl, whichmay be linear or branched.

In one or more embodiments, R₁ is a substituted alkyl, preferably asubstituted C₃₋₁₀ alkyl, preferably a substituted C₄₋₉ alkyl, preferablya substituted C₅₋₈ alkyl, preferably a substituted C₆₋₇ alkyl. Thecarbon counts described herein refers to a number of carbon atoms of thealkyl group of R₁ which excludes the carbon atoms of optionally presentsubstituents. The alkyl of R₁ may be substituted with at least onesubstituent selected from the group consisting of an alkyloxy, acycloalkyloxy, and an aryloxy, for example, methoxy, ethoxy, isopropoxy,cyclopentyloxy, cyclohexyloxy, and phenoxy. Alternatively, the alkyl ofR₁ may be substituted with an amine and/or an amide. The alkyl of R₁ maybe preferably substituted with an amide group such as acetamide,propanamide, and isobutyramide. In a preferred embodiment, the alkyl ofR₁ is substituted with acetamide.

In an alternative embodiment, R₁ is an unsubstituted alkyl, preferably alinear alkyl, preferably a linear C₃₋₁₀ alkyl, preferably a linear C₄₋₉alkyl, preferably a linear C₅₋₈ alkyl, preferably a linear C₆₋₇ alkyl.Exemplary linear alkyls include, but are not limited to n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl.Alternatively, R₁ is a branched alkyl, such as 6-methylheptyl,3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylhexyl,3,7-dimethyloctyl, and 2-propylheptyl. In a preferred embodiment, R₁ is6-methylheptyl, or 3-methylpentyl.

In one or more embodiments, R₁ is at least one selected from the groupconsisting of n-pentyl, n-hexyl, n-heptyl, 2-(cyclohexyloxy)ethyl,3-(cyclohexyloxy)propyl, 2-phenoxyethyl, 3-phenoxypropyl,4-isopropoxybutyl, 6-methylheptyl, 3-methylpentyl and 2-acetamidoethyl.In a preferred embodiment, R₁ is n-pentyl. In another preferredembodiment, R₁ is n-hexyl. In a most preferred embodiment, R₁ isn-pentyl.

As used herein, the value of m denotes an alkyl chain of —CH₂— groupsconnected between guanidino and amide groups of the compound of formula(I). In one or more embodiments, m is an integer in a range of 2-9,preferably 3-8, preferably 4-7, preferably 5-6. Most preferably, m is 4.

In some embodiments, the compound of formula (I) is one or more of thefollowing structures:

In a most preferred embodiment, the compound of formula (I) is

According to a second aspect, the present disclosure relates to acompound of formula (II)

or a salt thereof, a solvate thereof, a tautomer thereof, a stereoisomerthereof, or a mixture thereof.

R₂ is selected from the group consisting of an optionally substitutedamide, an optionally substituted aryl, and an optionally substitutedheteroaryl.

In one or more embodiments, R₂ is a monosubstituted alkylamide,preferably a monosubstituted C₂₋₅ alkylamide, preferably amonosubstituted C₃₋₄ alkylamide, such as N-methylamide (—C(O)NHCH₃),N-ethylamide (—C(O)NHC₂H₅), and N-(n-propyl)amide (—C(O)NHC₃H₇). Mostpreferably, R₂ is N-methylamide. In another embodiment, R₂ is anunsubstituted amide (—C(O)NH₂).

Alternatively, R₂ is an optionally substituted heteroaryl. In apreferred embodiment, R₂ is an optionally substituted pyridyl, or anoptionally substituted furyl. Most preferably, R₂ is a pyridyl, or afuryl.

As used herein, the value of n denotes an alkyl chain of —CH₂— groupsconnected between —R₂ and amide groups of the compound of formula (II).In one or more embodiments, n is an integer in a range of 1-4, or 2-3.In a preferred embodiment, n is 1.

R₃ is selected from the group consisting of an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, an optionally substituted alkenyl, and an optionallysubstituted aryl.

In one embodiment, R₂ is a monosubstituted alkylamide, and R₃ is asubstituted alkyl, preferably a substituted C₃₋₁₀ alkyl, preferably asubstituted C₄₋₉ alkyl, preferably a substituted C₅₋₈ alkyl, preferablya substituted C₆₋₇ alkyl. The alkyl of R₃ may be substituted with atleast one substituent selected from the group consisting of an alkyl, analkyloxy, a cycloalkyloxy, and an aryloxy, for example, methoxy, ethoxy,isopropoxy, cyclopentyloxy, cyclohexyloxy, and phenoxy. Alternatively,R₂ is a monosubstituted alkylamide, and R₃ is a linear (i.e.unsubstituted) alkyl, preferably a linear C₃₋₁₀ alkyl, preferably alinear C₄₋₉ alkyl, preferably a linear C₅₋₈ alkyl, preferably a linearC₆₋₇ alkyl. Exemplary linear alkyls include, but are not limited ton-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl,n-decyl. In a preferred embodiment, the linear alkyl is n-pentyl,n-hexyl, or n-heptyl. In another embodiment, R₂ is an unsubstitutedamide (—C(O)NH₂), and R₃ is a substituted alkyl, preferably asubstituted C₃₋₁₀ alkyl, preferably a substituted C₄₋₉ alkyl, preferablya substituted C₅₋₈ alkyl, preferably a substituted C₆₋₇ alkyl, mostpreferably 6-methylheptyl, or 3-methylpentyl. Alternatively, R₂ is amonosubstituted alkylamide, and R₃ is an optionally substituted alkenyl,preferably R₃ is a substituted C₅₋₁₆ alkenyl, preferably a substitutedC₆₋₁₄ alkenyl, preferably a substituted C₇₋₁₂ alkenyl, preferably asubstituted C₈₋₁₀ alkenyl. Most preferably, R₃ is3,7-dimethylocta-2,6-dien-1-yl.

In another embodiment, R₂ is a heteraryl, and R₃ is a substituted alkyl,preferably a substituted C₃₋₁₀ alkyl, preferably a substituted C₄₋₉alkyl, preferably a substituted C₅₋₈ alkyl, preferably a substitutedC₆₋₇ alkyl. The alkyl of R₃ may be substituted with an amide group.Exemplary amide groups include, but are not limited to, acetamide,propanamide, and isobutyramide. In a preferred embodiment, the alkyl ofR₃ is substituted with acetamide. Alternatively, R₂ is a heteraryl, andR₃ is a linear (i.e. unsubstituted) alkyl, preferably a linear C₃₋₁₀alkyl, preferably a linear C₄₋₉ alkyl, preferably a linear C₅₋₈ alkyl,preferably a linear C₆₋₇ alkyl. Exemplary linear alkyls include, but arenot limited to n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl. In a preferred embodiment, the linear alkyl isn-hexyl.

In at least one embodiment, R₃ is selected from the group consisting ofn-pentyl, n-hexyl, n-heptyl, 2-(cyclohexyloxy)ethyl,3-(cyclohexyloxy)propyl, 2-phenoxyethyl, 3-phenoxypropyl,4-isopropoxybutyl, 6-methylheptyl, 3-methylpentyl, 2-acetamidoethyl, and3,7-dimethylocta-2,6-dien-1-yl.

In some embodiments, the compound of formula (II) has one of thefollowing structures:

The compounds of the present disclosure may be prepared by methods knownto those of ordinary skills in the art. The following methods set forthbelow are provided for illustrative purposes and are not intended tolimit the scope of the disclosure. It will be recognized that it may bepreferred or necessary to prepare such a compound in which a functionalgroup is protected using a conventional protecting group then to removethe protecting group to provide a compound of the present disclosure.

The compounds of formulae (I) and (II) may, for example, be synthesizedaccording to a process illustrated in FIG. 2. Briefly, a 2,4-disubstituted imidazole (i.e. 2-(ethoxycarbonyl)-1H-imidazole-4-carboxylicacid (MOC-02)) may be formed via (i) a cyclization reaction between anoxime (i.e. (Z)-2-amino-2-(hydroxyimino) acetate) and an alkyne (i.e.tert-butyl propiolate) thereby forming a disubstituted imidazoletert-butyl ester (i.e. 5-(tert-butyl) 2-ethyl1H-imidazole-2,5-dicarboxylate (MOC-01)), and (ii) deprotection oftert-butyl ester group of MOC-01 thereby forming MOC-02 [Duguay, G.;Guémas, J.-P.; Meslin, J.-C.; Pradere, J.-P.; Reliquet, F.; Reliquet,A.; Tea-Gokou, C.; Quiniou, H.; Rabiller, C., Heteroatomic chains andtheir products of cyclisation. IV.t-butyl-2-phthalimido-2-(3,6-dihydro-1,3-2H-thiazine-2-yliden)-acetatessubstituted in position 5 by a functional group. Journal of HeterocyclicChemistry 1980, 17 (4), 767-770; and Vuilhorgne, M.; Malpart, J.; Mutti,S.; Mignani, S., Preparative route to2-ethoxycarbonylimidazole-4-phosphonate anddiethylimidazole-2,4-dicarboxylate. Journal of Heterocyclic Chemistry2003, 40 (1), 159-162, each incorporated herein by reference in theirentirety]. Alternatively, MOC-02 may be available from commercialvendors including, without limitation, Accel Pharmtech, Asta Tech,Ambeed, Alichem, BLD Pharmatech, and FCH group.

In one embodiment, the guanidino side chain of compound of formula (I)may be incorporated via amidation reaction between the carboxylic groupof MOC-02 and an amine of formula (III)

wherein m is as previously specified, and R_(p) is a protecting groupintended to avoid formation of undesired bonds during the amidation. Thedetails concerning the use of protecting groups in accordance with thepresent invention are known to those of ordinary skills in the art.Protecting groups that can be used are listed, for example, in Greene,et al., “Protective Groups in Organic Synthesis”, Wiley-Interscience,1999, hereby incorporated by reference in its entirety. Exemplaryprotecting groups include, but are not limited to, acyl groups such asformyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl, aromaticcarbamate groups such as benzyloxycarbonyl (Cbz or Z), and substitutedbensyloxycarbonyls, and 9-fluorenylmethyloxycarbonyl (Fmoc), aliphaticcarbamate groups such as tert-butyloxycarbonyl (Boc), ethoxycarbonyl,diisopropylmethoxycarbonyl, and allyloxycarbonyl, cyclic alkyl carbamategroups such as cyclopentyloxycarbonyl and adamantyloxycarbonyl, alkylgroups such as triphenylmethyl and benzyl, trialkylsilyl such astrimethylsilyl, and thiol containing groups such as phenylthiocarbonyl,and dithiasuccinoyl. In a preferred embodiment, R_(p) of the amine offormula (III) is Boc. After the first amidation reaction, a guanidinocompound of formula (IV) may be obtained

The protecting group R, may be cleaved after the amidation reactionusing deprotection protocols generally known to those of ordinary skillin the art. For example, when the Boc group is used, the methods ofchoice may be trifluoroacetic acid, neat or in dichioromethane, or HClin dioxane or in ethyl acetate.

In another embodiment, the R₂ amide side chain of the compound offormula (II) may be incorporated via a first amidation reaction betweenthe carboxylic group of MOC-02 and an amine of formula (V):

wherein R₂ and n are as previously specified. After the first amidationreaction, a compound of formula (VI) may be obtained

Generally, the first amidation reaction may be performed in a solventsuch as tetrahydrofuran, benzene, xylene, dimethylformamide, ethylacetate, diethyl ether, acetonitrile, dimethyl sulfoxide, methylenechloride, chloroform, nitrobenzene, isopropanol, and mixtures thereof.Preferably, tetrahydrofuran is used as the solvent. In a preferredembodiment, a molar ratio of the amine of formula (III) or (V) to thedisubstituted imidazole (i.e. MOC-02) is in a range of 1:1 to 3:1,preferably 1.05:1 to 2:1, preferably 1.1:1 to 1.5:1, or about 1.2:1.

In preferred embodiments, a base such as trimethylamine, trimethylamine,diisopropylethylamine (DIPEA), triisopropylamine,dimethylaminopropylamine, N-methylmorpholine, N-methylpyrrolidine,4-dimethylaminopyridine (DMAP), and 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) is present in the first amidation reaction. Preferably, the baseis DIPEA. In a preferred embodiment, a molar ratio of the base to thedisubstituted imidazole (i.e. MOC-02) is in a range of 1:1 to 5:1,preferably 1.2:1 to 3:1, preferably 1.5:1 to 2:1, or about 1.75:1.

In a related embodiment, amide bond formation coupling reagents may beused to facilitate and/or accelerate the first amidation reaction.Examples of such coupling reagents include, without limitation,carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCor EDCI), N,N-dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIC), 1H-benzotriazole derivatives such as1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), O-benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU), andN-[(5-chloro-3-oxido-1H-benzotriazol-1-yl)-4-morpholinylmethylene]-N-methylmethanaminiumhexafluorophosphate (HDMC), and carbonyldiimidazole (CDI).

In another related embodiment, an additive that minimizes racemizationduring the amidation reaction may be used in combination of theaforementioned coupling reagent. Exemplary additives include, but arenot limited to, 1-hydroxy-7-azabenzotriazole (HOAt),hydroxybenzotriazole (HOBt), 6-chloro-1-hydroxybenzotriazole (6-ClHOBt),ethyl 1-hydroxy-1H-1,2,3-triazole-4-carboxylate (HOCt),1-hydroxy-2-pyridinone (HOPy), ethyl 2-cyano-2-(hydroxyimino)acetate(Oxyma), N-hydroxy-5-norbornene2,3-dicarboximide (HONB),N-hydroxysuccinimide (HOSu),3-hydroxy-4-oxo-3,4-dihydro1,2,3-benzotriazine (HODhbt), and copper(I)iodide. Preferably, the additive is HOBt.

In a most preferred embodiment, the coupling reagent is EDCI and theadditive is HOBt. A molar ratio of the coupling reagent (e.g. EDCI) tothe disubstituted imidazole (i.e. MOC-02) is in a range of 1:1 to 3:1,preferably 1.2:1 to 2:1, or about 1.5:1. A molar ratio of the couplingreagent (e.g. EDCI) to the additive (e.g. HOBt) is in a range of 1:2 to2:1, preferably 1:1.5 to 1.5:1, or about 1:1.

In a preferred embodiment, the base, the coupling reagent, and theadditive are each introduced to the first amidation reaction mixture ina two-stage or multi-stage fashion. For example, a first portion of thebase, the coupling reagent, or the additive which is 50-75%, 55-70%, or57-67% of a total mole of the base, the coupling reagent, or theadditive used herein may be added to the reaction mixture and allowed toreact for 1-5 hours, 2-4 hours, or about 3 hours, and subsequently asecond portion of the base, the coupling reagent, or the additive whichis 25-50%, 30-45%, or 33-43% of a total mole of the base, the couplingreagent, or the additive used herein may be added to the same reactionmixture. Alternatively, the base, the coupling reagent, and the additivemay be introduced to the reaction mixture in one batch. In one or moreembodiments, the first amidation reaction mixture is reacted at atemperature of 20-80° C., preferably 30-70° C., preferably 40-60° C.under agitation. The first amidation reaction may be conducted in inertgas (e.g. nitrogen, argon, helium). Also, in some embodiments, thereaction may not be conducted in inert gas, but in a vacuum.

Methods of agitation include, without limitation, using an agitator, avortexer, a rotary shaker, a magnetic stirrer, a centrifugal mixer, oran overhead stirrer. In another embodiment, the mixture and/or thereaction mixture is left to stand (i.e. not stirred). Alternatively, themixture and/or the reaction mixture is sonicated in an ultrasonic bathor with an ultrasonic probe.

The compound of formula (I) may be produced via a second amidationreaction. In one embodiment, the amidation reaction involves (i) addinga base to the compound of formula (IV) to form a carboxylate salt, and(ii) reacting the carboxylate salt with a proper amine of formula (VII)H₂N—R₁,  (VII)thereby forming the compound of formula (I), wherein R₁ is as previouslyspecified.

The compound of formula (II) may be synthesized similarly via a secondamidation reaction between the compound of formula (VI) and a properamine of formula (VIII)H₂N—R₃,  (VIII)wherein R₃ is as previously described.

The second amidation reaction may be performed in a solvent such astetrahydrofuran, benzene, xylene, dimethylformamide, ethyl acetate,diethyl ether, acetonitrile, dimethyl sulfoxide, methylene chloride,chloroform, nitrobenzene, isopropanol, and mixtures thereof. Preferably,tetrahydrofuran is used as the solvent for the second amidation. In apreferred embodiment, a molar ratio of the amine of formula (VII) to thecompound of formula (IV) is in a range of 0.9:1 to 2:1, preferably 1:1to 1.5:1, or about 1.2:1.

Exemplary bases used herein for the second amidation include, withoutlimitation, inorganic bases such as lithium hydroxide, sodium hydroxide,potassium hydroxide, rubidium hydroxide, and cesium hydroxide, andorganic bases such as trimethylamine, trimethylamine,diisopropylethylamine (DIPEA), triisopropylamine,dimethylaminopropylamine, N-methylmorpholine, N-methylpyrrolidine,4-dimethylaminopyridine (DMAP), 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU), and mixtures thereof. Preferably, an inorganic base is usedherein for the second amidation. More preferably, lithium hydroxide isused as the base. In one embodiment, a molar ratio of the base to thecompound of formula (IV) is in a range of 1:1 to 6:1, preferably 2:1 to5:1, more preferably 3:1 to 4:1.

A coupling reagent may be added to the reaction mixture of thecarboxylate salt and the amine of formula (VII) to facilitate the secondamidation reaction. Preferably, the coupling reagent is aphosphorus-based coupling reagent. Exemplary phosphorus-based couplingreagents include, but are not limited to, propylphosphonic anhydride(T3P®), diphenylphosphorazidate (DPPA),diethyl-2-(3-oxo-2,3-dihydro-1,2-benzisosulfonazolyl)phosphonate (DEBP),N,N′-bismorpholinophosphonic chloride (BMPCl), 1-oxo-chlorophospholane(CptCl), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one diethyltartarate (DEPBT), phosphoric acid3,5-dioxo-10-oxa-4-azatricyclo[5.2.1.0]dec-8-en-4-yl ester diphenylester (ENDPP), norborn-5-ene-2,3-dicarboximidodiphenylphosphate (NDPP),and bis(2-oxooxazolidin-3-yl)phosphinic chloride (BopCl). In a preferredembodiment, T3P® is used herein as the coupling reagent. Alternatively,the coupling reagents and additives described previously for the firstamidation reaction may be used in addition to, or in lieu of thephosphorus-based coupling reagent.

In one embodiment, a molar ratio of the phosphorus-based couplingreagent to the amine of formula (VII) is in a range of 4:1 to 20:1,preferably 6:1 to 16:1, preferably 8:1 to 12:1, or about 10:1.Preferably, the phosphorus-based coupling reagent may be introduced tothe second amidation reaction in a two-stage or multi-stage fashion.Alternatively, the phosphorus-based coupling reagent may be introducedto the reaction mixture in one batch. In one or more embodiments, thesecond amidation reaction is performed at a temperature of 4-40° C.,preferably 10-30° C., preferably 15-25° C. under agitation. The secondamidation reaction may be conducted in inert gas (e.g. nitrogen, argon,helium). Also, in some embodiments, the reaction may not be conducted ininert gas, but in a vacuum.

The compound of formulae (I) and (II) may be isolated and purified bymethods known to those of ordinary skill in the art, such ascrystallization, filtration through a celite containing cartridge,evaporating the reaction mixture to dryness, aqueous work-up, extractionwith organic solvents, distillation, column chromatography, and highpressure liquid chromatography (HPLC) on normal phase or reversed phase.Preferred methods include column chromatography and recrystallization.

Drug research and development efforts made by global researchinstitutions and pharmaceutical industries have been focused on otherHCV targets rather than NS4A. NS4A binding pocket remained anunder-explored target for discovering new antiviral agents, which maypossibly overcome emerging resistance against available drugs.Structurally, NS4A is a small peptide with 54 amino acids, which areinclined of forming proper assembly with other viral proteins. Forinstance, the hydrophobic N-terminal initiates a sandwich-fillingbinding that brings the N-terminal (A₀ and A₁ sheets) of the NS3 torearrange and conform properly. This process is critical for theenzyme's catalytic site to attain a suitable shape that accommodates thepeptide substrate [De Francesco, R.; Tomei, L.; Altamura, S.; Summa, V.;Migliaccio, G., Approaching a new era for hepatitis C virus therapy:inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNApolymerase. Antiviral Res 2003, 58 (1), 1-16; Ishido, S.; Fujita, T.;Hotta, H., Complex formation of NS5B with NS3 and NS4A proteins ofhepatitis C virus. Biochemical and biophysical research communications1998, 244 (1), 35-40; Failla, C.; Tomei, L.; De Francesco, R., Both NS3and NS4A are required for proteolytic processing of hepatitis C virusnonstructural proteins. Journal of virology 1994, 68 (6), 3753-3760; andHamad, H. A.; Thurston, J.; Teague, T.; Ackad, E.; Yousef, M. S., TheNS4A cofactor dependent enhancement of HCV NS3 protease activitycorrelates with a 4D geometrical measure of the catalytic triad region.PloS one 2016, 11 (12), e0168002]. It was found that the central part ofNS4A (Gy21-Leu34) with only 14 amino acids is required for theactivation of NS3 protease. Shimizu et al. observed inhibition of theenzyme via mutating the basic Arg-28 residue to a neutral glutamine(Gln) [Shimizu, Y.; Yamaji, K.; Masuho, Y.; Yokota, T.; Inoue, H.; Sudo,K.; Satoh, S.; Shimotohno, K., Identification of the sequence on NS4Arequired for enhanced cleavage of the NS5A/5B site by hepatitis C virusNS3 protease. Journal of virology 1996, 70 (1), 127-132]. Other worksthat discussed the NS4A site inhibitors are either presumptive [DeFrancesco, R.; Pessi, A.; Steinkihler, C., Mechanisms of hepatitis Cvirus NS3 proteinase inhibitors. Journal of viral hepatitis 1999, 6,23-30; and Kim, J.; Morgenstern, K.; Lin, C.; Fox, T.; Dwyer, M.;Landro, J.; Chambers, S.; Markland, W.; Lepre, C.; O'malley, E., Crystalstructure of the hepatitis C virus NS3 protease domain complexed with asynthetic NS4A cofactor peptide. Cell 1996, 87 (2), 343-355] or virtual(i.e. computational modeling) [Hamad, H. A.; Thurston, J.; Teague, T.;Ackad, E.; Yousef, M. S., The NS4A cofactor dependent enhancement of HCVNS3 protease activity correlates with a 4D geometrical measure of thecatalytic triad region. PloS one 2016, 11 (12), e0168002]. As a result,non-peptide competitors of NS4A central part may be viable therapeuticoptions in DAA class of HCV therapeutics.

After careful inspection of 3D-visualization of crystal structures ofNS4A in bound form, it was found that it forms a β-sheet that is mostlyextended except a turn featuring a nearly planar area composed of aneclipsed cis bond and four trans bonds with the backbone that spanthrough Val-26 to Arg-28. This turn is facilitated by the presence ofthe Gly-27 due to its small size (no side chains) (see FIG. 1).Accordingly, the compounds of formulae (I) and (II) disclosed herein areproposed via a De novo structure-based design by deriving the planararea into imidazole nucleus. Similar imidazole cores are found inseveral commercialized drugs such as nitronidazole (antimicrobial),azathioprine (immunosuppressive), nilotinib (anticancer), and manyothers [Zhang, L.; Peng, X. M.; Damu, G. L.; Geng, R. X.; Zhou, C. H.,Comprehensive review in current developments of imidazole-basedmedicinal chemistry. Med Res Rev 2014, 34 (2), 340-437].

According to a third aspect, the present disclosure relates to apharmaceutical composition involving the compound of formula (I) of thefirst aspect and a pharmaceutically acceptable carrier and/or excipient.

In one or more embodiments, the compound of formula (I) is

In certain embodiments, the compound of formula (II) is present inaddition to, or in lieu of the compound of formula (I) in thepharmaceutical composition.

As used herein, a “composition” or a “pharmaceutical composition” refersto a mixture of the active ingredient with other chemical components,such as pharmaceutically acceptable carriers and excipients. One purposeof a composition is to facilitate administration of the compounddisclosed herein in any of its embodiments to a subject. Pharmaceuticalcompositions of the present disclosure may be manufactured by processeswell known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Depending on theintended mode of administration (oral, parenteral, or topical), thecomposition can be in the form of solid, semi-solid or liquid dosageforms, such as tablets, suppositories, pills, capsules, powders,liquids, or suspensions, preferably in unit dosage form suitable forsingle administration of a precise dosage.

The term “active ingredient”, as used herein, refers to an ingredient inthe composition that is biologically active, for example, the compoundrepresented by formula (I), the compound represented by formula (II), asalt thereof, a solvate thereof, a tautomer thereof, a stereoisomerthereof, or any mixtures thereof. In some embodiments, other activeingredients in addition to the compound of the current disclosure may beincorporated into a pharmaceutical composition.

In one or more embodiments, the pharmaceutical composition disclosedherein further includes an antiviral agent that is structurally distinctfrom the compounds of formulae (I) and (II). In one embodiment, theantiviral agent used herein does not have anti-HCV activity. Preferably,the antiviral agent has anti-HCV activity. As used herein, the term“anti-HCV activity” means the agent is effective to inhibit the functionof at least one target selected from the group consisting of HCVmetalloprotease, HCV serine protease, HCV polymerase, HCV helicase, HCVNS4B protein, HCV entry, HCV assembly, HCV egress, HCV NS5A protein,IMPDH and a nucleoside analog for the treatment of an HCV infection. Inone embodiment, the antiviral agent having anti-HCV activity iseffective to inhibit the function of a target in the HCV life cycleother than the HCV serine protease.

Exemplary antiviral agents with anti-HCV activity include, but are notlimited to, Imiqimod, ribavirin, amantadine, rimantadine, interferonsuch as interferon alpha 213, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, and lymphoblastiod interferon tau,interleukins such as interleukin 2, interleukin 6, and interleukin 12,other inhibitors of HCV NS3 protease, inhibitors of other targets in theHCV life cycle such as helicase, polymerase, metalloprotease, orinternal ribosome entry site; or combinations thereof, and the like.Other antiviral agents such as oseltamivir (Tamiflu), zanamivir,peramivir (Rapivab®) may be further combined into the pharmaceuticalcomposition for the prevention or treatment of secondary infection.Alternatively, the antiviral agent may act as an additional therapy, andthe compounds of formula (I) and/or (II) may be administered with theantiviral agent in combination therapy, either jointly or separately.

In one or more embodiments, the pharmaceutical composition comprises upto 0.01%, up to 0.1%, up to 1%, up to 5%, or up to 10% by weight of thepharmaceutically acceptable carrier and/or excipient relative to a totalweight of the pharmaceutical composition. In one or more embodiments,the pharmaceutical composition comprises at least 0.01 wt %, at least0.05 wt %, at least 0.1 wt %, at least 0.5 wt %, at least 5 wt %, atleast 10 wt %, at least 15 wt %, at least 20 wt %, at least 25 wt %, atleast 30 wt %, at least 35 wt %, at least 40 wt %, at least 45 wt %, atleast 50 wt %, at least 55 wt %, at least 60 wt %, at least 65 wt %, atleast 70 wt %, at least 75 wt %, at least 80 wt %, at least 85 wt %, atleast 90 wt %, at least 95 wt %, at least 99 wt %, or at least 99.9 wt %of the compound of formula (I) relative to a total weight of thepharmaceutical composition. The pharmaceutical composition may contain0.5-500 μM of the compound of formula (I) relative to a total volume ofthe composition, preferably 1-400 μM, preferably 10-300 μM, preferably20-200 μM of the compound of formula (I) relative to the total volume ofthe composition. In some embodiments, the composition comprises up to0.1 wt %, 1 wt %, 5 wt %, or 10 wt % of a pharmaceutically acceptablesalt of the compound of formula (I). In some embodiments, thecomposition comprises up to 0.1 wt %, 1 wt %, 5 wt %, or 10 wt % of apharmaceutically acceptable solvate of the compound of formula (I).Preferably, the composition may further comprise pharmaceuticallyacceptable binders, such as sucrose, lactose, xylitol, andpharmaceutically acceptable excipients such as calcium carbonate,calcium phosphate, and dimethyl sulfoxide (DMSO).

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism, does not abrogate the biological activity and properties ofthe administered active ingredient, and/or does not interact in adeleterious manner with the other components of the composition in whichit is contained. The term “carrier” encompasses any excipient, binder,diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or othermaterial well known in the art for use in pharmaceutical formulations.The choice of a carrier for use in a composition will depend upon theintended route of administration for the composition. The preparation ofpharmaceutically acceptable carriers and formulations containing thesematerials is described in, e.g. Remington's Pharmaceutical Sciences,21st Edition, ed. University of the Sciences in Philadelphia,Lippincott, Williams & Wilkins, Philadelphia Pa., 2005, which isincorporated herein by reference in its entirety). Examples ofphysiologically acceptable carriers include antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.;Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS™ (BASF;Florham Park, N.J.). An “excipient” refers to an inert substance addedto a composition to further facilitate administration of a compound.Examples, without limitation, of excipients include calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils, and polyethylene glycols.

In one or more embodiments, the pharmaceutically acceptable carrierand/or excipient is at least one selected from the group consisting of abuffer, an inorganic salt, a fatty acid, a vegetable oil, a syntheticfatty ester, a surfactant, and a polymer.

Exemplary buffers include, but are not limited to, phosphate buffers,citrate buffer, acetate buffers, borate buffers, carbonate buffers,bicarbonate buffers, and buffers with other organic acids and salts.

Exemplary inorganic salts include, but are not limited to, calciumcarbonate, calcium phosphate, disodium hydrogen phosphate, potassiumhydrogen phosphate, sodium chloride, zinc oxide, zinc sulfate, andmagnesium trisilicate.

Exemplary fatty acids include, but are not limited to, an omega-3 fattyacid (e.g., linolenic acid, docosahexaenoic acid, eicosapentaenoic acid)and an omega-6 fatty acid (e.g., linoleic acid, eicosadienoic acid,arachidonic acid). Other fatty acids, such as oleic acid, palmitoleicacid, palmitic acid, stearic acid, and myristic acid, may be included.

Exemplary vegetable oils include, but are not limited to, avocado oil,olive oil, palm oil, coconut oil, rapeseed oil, soybean oil, corn oil,sunflower oil, cottonseed oil, and peanut oil, grape seed oil, hazelnutoil, linseed oil, rice bran oil, safflower oil, sesame oil, brazil nutoil, carapa oil, passion fruit oil, and cocoa butter.

Exemplary synthetic fatty esters include, without limitation, methyl,ethyl, isopropyl and butyl esters of fatty acids (e.g., isopropylpalmitate, glyceryl stearate, ethyl oleate, isopropyl myristate,isopropyl isostearate, diisopropyl sebacate, ethyl stearate, di-n-butyladipate, dipropylene glycol pelargonate), C₁₂-C₁₆ fatty alcohol lactates(e.g., cetyl lactate and lauryl lactate), propylene dipelargonate,2-ethylhexyl isononoate, 2-ethylhexyl stearate, isopropyl lanolate,2-ethylhexyl salicylate, cetyl myristate, oleyl myristate, oleylstearate, oleyl oleate, hexyl laurate, isohexyl laurate, propyleneglycol fatty ester, and polyoxyethylene sorbitan fatty ester. As usedherein, the term “propylene glycol fatty ester” refers to a monoether ordiester, or mixtures thereof, formed between propylene glycol orpolypropylene glycol and a fatty acid. The term “polyoxyethylenesorbitan fatty ester” denotes oleate esters of sorbitol and itsanhydrides, typically copolymerized with ethylene oxide.

Surfactants may act as detergents, wetting agents, emulsifiers, foamingagents, and dispersants. Surfactants that may be present in thecompositions of the present disclosure include zwitterionic (amphoteric)surfactants, e.g., phosphatidylcholine, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),anionic surfactants, e.g., sodium lauryl sulfate, sodium octanesulfonate, sodium decane sulfonate, and sodium dodecane sulfonate,non-ionic surfactants, e.g., sorbitan monolaurate, sorbitanmonopalmitate, sorbitan trioleate, polysorbates such as polysorbate 20(Tween 20), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80),cationic surfactants, e.g., decyltrimethylammonium bromide,dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,tetradecyltrimethyl-ammonium chloride, and dodecylammonium chloride, andcombinations thereof.

Exemplary polymers include, without limitation, polylactides,polyglycolides, polycaprolactones, polyanhydrides, polyurethanes,polyesteramides, polyorthoesters, polydioxanones, polyacetals,polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), poly(maleic anhydride), apolyvinyl alcohols, and copolymers, terpolymers, or combinations ormixtures therein. The copolymer/terpolymer may be a randomcopolymer/terpolymer, or a block copolymer/terpolymer.

Depending on the route of administration e.g. oral, parental, ortopical, the composition may be in the form of solid dosage form such astablets, caplets, capsules, powders, and granules, semi-solid dosageform such as ointments, creams, lotions, gels, pastes, andsuppositories, liquid dosage forms such as solutions, and dispersions,inhalation dosage form such as aerosols, and spray, or transdermaldosage form such as patches.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive ingredient is ordinarily combined with one or more adjuvantsappropriate to the indicated route of administration. If administeredper os, the active ingredient can be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, cellulose alkylesters, talc, stearic acid, magnesium stearate, magnesium oxide, sodiumand calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering ingredients such as sodium citrate,magnesium or calcium carbonate or bicarbonate. Tablets and pills canadditionally be prepared with enteric coatings.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting ingredients,emulsifying and suspending ingredients, and sweetening, flavouring, andperfuming ingredients.

For therapeutic purposes, formulations for parenteral administration canbe in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. The term “parenteral”, as used herein,includes intravenous, intravesical, intraperitoneal, subcutaneous,intramuscular, intralesional, intracranial, intrapulmonal, intracardial,intrasternal, and sublingual injections, or infusion techniques. Thesesolutions and suspensions can be prepared from sterile powders orgranules having one or more of the carriers or diluents mentioned foruse in the formulations for oral administration. The active ingredientcan be dissolved in water, polyethylene glycol, propylene glycol,ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzylalcohol, sodium chloride, and/or various buffers. Other adjuvants andmodes of administration are well and widely known in the pharmaceuticalart.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting ingredients and suspendingingredients. The sterile injectable preparation can also be a sterileinjectable solution or suspension in a non-toxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employedincluding synthetic mono- or di-glycerides. In addition, fatty acids,such as oleic acid, find use in the preparation of injectables. Dimethylacetamide, surfactants including ionic and non-ionic detergents,polyethylene glycols can be used. Mixtures of solvents and wettingingredients such as those discussed above are also useful.

Suppositories for rectal administration can be prepared by mixing theactive ingredient with a suitable non-irritating excipient, such ascocoa butter, synthetic mono-, di-, or triglycerides, fatty acids, andpolyethylene glycols that are solid at ordinary temperatures but liquidat the rectal temperature and will therefore melt in the rectum andrelease the drug.

Topical administration may involve the use of transdermal administrationsuch as transdermal patches or iontophoresis devices. Formulation ofdrugs is discussed in, for example, Hoover, J. E. Remington'spharmaceutical sciences, Mack Publishing Co., Easton, Pa., 1975; andLiberman, H. A.; Lachman, L., Eds. Pharmaceutical dosage forms, MarcelDecker, New York, N.Y., 1980, which are incorporated herein by referencein their entirety.

In other embodiments, the pharmaceutical composition having the compoundof formula (I), the salt thereof, the solvate thereof, the tautomerthereof, the stereoisomer thereof, or the mixture thereof has differentrelease rates categorized as immediate release and controlled- orsustained-release.

As used herein, immediate release refers to the release of an activeingredient substantially immediately upon administration. In anotherembodiment, immediate release occurs when there is dissolution of anactive ingredient within 1-20 minutes after administration. Dissolutioncan be of all or less than all (e.g. about 70%, about 75%, about 80%,about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, 99.9%, or99.99%) of the active ingredient. In another embodiment, immediaterelease results in complete or less than complete dissolution withinabout 1 hour following administration. Dissolution can be in a subject'sstomach and/or intestine. In one embodiment, immediate release resultsin dissolution of an active ingredient within 1-20 minutes afterentering the stomach. For example, dissolution of 100% of an activeingredient can occur in the prescribed time. In another embodiment,immediate release results in complete or less than complete dissolutionwithin about 1 hour following rectal administration. In someembodiments, immediate release is through inhalation, such thatdissolution occurs in a subject's lungs.

Controlled-release, or sustained-release, refers to a release of anactive ingredient from a composition or dosage form in which the activeingredient is released over an extended period of time. In oneembodiment, controlled-release results in dissolution of an activeingredient within 20-180 minutes after entering the stomach. In anotherembodiment, controlled-release occurs when there is dissolution of anactive ingredient within 20-180 minutes after being swallowed. Inanother embodiment, controlled-release occurs when there is dissolutionof an active ingredient within 20-180 minutes after entering theintestine. In another embodiment, controlled-release results insubstantially complete dissolution after at least 1 hour followingadministration. In another embodiment, controlled-release results insubstantially complete dissolution after at least 1 hour following oraladministration. In another embodiment, controlled-release results insubstantially complete dissolution after at least 1 hour followingrectal administration. In one embodiment, the pharmaceutical compositiondescribed herein is not a controlled-release composition.

According to a fourth aspect, the present disclosure relates to a methodof preventing or treating hepatitis C virus (HCV) infection. The methodinvolves administering the pharmaceutical composition of the thirdaspect to a subject in need of therapy.

As used herein, the term “preventing” in the context of theadministration of a therapy to a subject in need thereof refers topreventing a disease, disorder or condition from occurring in a subjectwhich may be predisposed to the disease, disorder and/or condition buthas not yet been diagnosed as having it. The term “treating” refers to(i) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and (ii) relieving the disease, disorder or condition,i.e., causing regression of the disease, disorder and/or condition.Treatment is preferably commenced at the time of infection or postinfection with HCV. It is recommended that the treatment continues untilthe virus is no longer present or active. For protecting a non-infectedsubject from future infection, the treatment continues for as long asthere is a potential exposure to the virus.

The term “subject” and “patient” are used interchangeably. As usedherein, they refer to any subject for whom or which therapy, includingwith the compositions according to the present disclosure is desired. Inmost embodiments, the subject is a mammal, including but not limited toa human, a non-human primate such as a chimpanzee, a domestic livestocksuch as a cattle, a horse, a swine, a pet animal such as a dog, a cat,and a rabbit, and a laboratory subject such as a rodent, e.g. a rat, amouse, and a guinea pig. In preferred embodiments, the subject is ahuman.

The terms “administer”, “administering”, “administration”, and the like,as used herein, refer to the methods that may be used to enable deliveryof the active ingredient and/or the composition to the desired site ofbiological action. Routes or modes of administration are as set forthherein. These methods include, but are not limited to, oral routes,intraduodenal routes, parenteral injection (including intravenous,subcutaneous, intraperitoneal, intramuscular, intravascular, orinfusion), topical and rectal administration. Those of ordinary skill inthe art are familiar with administration techniques that can be employedwith the compounds and methods described herein. In a preferredembodiment, the active ingredient and/or the pharmaceutical compositiondescribed herein are administered orally.

In one or more embodiments, the pharmaceutical composition administeredcomprises the compound of formula (I), or a salt thereof, a solvatethereof, a tautomer thereof, a stereoisomer thereof, or a mixturethereof. In a preferred embodiment, the pharmaceutical compositionadministered comprises a compound which is

The dosage amount and treatment duration are dependent on factors, suchas bioavailability of a drug, administration mode, toxicity of a drug,gender, age, lifestyle, body weight, the use of other drugs and dietarysupplements, the disease stage, tolerance and resistance of the body tothe administered drug, etc., and then determined and adjustedaccordingly. The terms “effective amount”, “therapeutically effectiveamount”, or “pharmaceutically effective amount” refer to that amount ofthe active ingredient being administered which will relieve to someextent one or more of the symptoms of the disease being treated. Theresult can be a reduction and/or alleviation of the signs, symptoms, orcauses of a disease, or any other desired alteration of a biologicalsystem. An appropriate “effective amount” may differ from one individualto another. An appropriate “effective amount” in any individual case maybe determined using techniques, such as a dose escalation study. In oneor more embodiments, an effective amount of the compound of formula (I)in a range of 0.1-100 mg/kg, preferably 0.5-50 mg/kg, more preferably1-25 mg/kg is administered per body weight of the subject. However, incertain embodiments, the effective amount of the compound of formula (I)is less than 0.1 mg/kg or greater than 100 mg/kg. In general, thecompound is most desirably administered at a concentration level thatwill generally afford antivirally effective results without causing anyharmful or deleterious side effects.

A treatment method may comprise administering a pharmaceuticalcomposition containing the compound of formula (I) of the currentdisclosure in any of its embodiments as a single dose or multipleindividual divided doses. In some embodiments, the interval of timebetween the administration of the composition and the administration ofone or more additional therapies (e.g. interferon, ribavirin) may beabout 1-5 minutes, 1-30 minutes, 30 minutes to 60 minutes, 1 hour, 1-2hours, 2-6 hours, 2-12 hours, 12-24 hours, 1-2 days, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks,26 weeks, 52 weeks, 11-15 weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks,40-50 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months,7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2years, or any period of time in between. Preferably, the pharmaceuticalcompositions disclosed herein will be administered from about 1 to about5 times per day or alternatively, as a continuous infusion for at least2 days, at least 5 days, at least 6 days, or at least 7 days. Suchadministration can be used as a chronic or acute therapy. In certainembodiments, the composition and one or more additional therapies areadministered less than 1 day, less than 1 week, less than 2 weeks, lessthan 3 weeks, less than 4 weeks, less than 1 month, less than 2 months,less than 3 months, less than 6 months, less than 1 year, less than 2years, or less than 5 years apart.

The pharmaceutical composition may be administered in vivo to thesubject to inhibit HCV NS3 protease or to treat or prevent HCV virusinfection. In one or more embodiments, the compound of formula (I) ofthe current disclosure in any of its embodiments binds to an activationsite of hepatitis C virus (HCV) serine protease. As used herein, the“serine protease” is referred to as NS3 protease. The compound offormula (I) may preferably bind to the activation site of HCV NS3protease such as but not limited to linear peptides, cyclic peptides,macrolactons, macrolactams, and peptidomimetics. In a preferredembodiment, the compound of formula (I) of the current disclosure in anyof its embodiments binds to NS3/4A serine protease.

As used herein, “bind” or “binding” refers to a process involving anactive ingredient (e.g. the compound of formula (I)) reversibly orirreversibly binds to HCV NS3 protease or variant thereof at theactivation site. Binding may involve the formation of bonds which may becovalent or non-covalent. Non-covalent bonds may be e.g. hydrogen bonds,ionic bonds, or hydrophobic interactions. The active ingredient (e.g.the compound of formula (I)) is expected to interfere and inhibit theinteractions leading to the formation of the active form of the NS3protease. Binding can be quantitated in accordance with methodswell-known in the art and described herein below.

In certain embodiments, a suitable assay is used to characterize thepotential binding ability of the active ingredient (e.g. compound offormula (I)) to bind to the activation site. This may involve directlytesting the active ingredient's ability to bind, and/or determiningwhether the active ingredient has an influence on the binding of theNS4A to HCV NS3 protease or variants thereof. To evaluate bindingproperties of binding compounds, assays may be used. Exemplary assaymethods include, but not limited to, calorimetric techniques, surfaceplasmon resonance (SPR), and spectroscopic methods such as NMR,fluorescence, and UV-vis spectroscopies.

Calorimetric methods include but not limited to isothermal titrationcalorimetry and differential scanning calorimetry. SPR is the resonantoscillation of conduction electrons at the interface between negativeand positive permittivity material stimulated by incident light. Themethod involves immobilizing one molecule of a binding pair on thesensor chip surface (“ligand”, in Biacore parlance) and injecting aseries of concentrations of its partner (“analyte”) across the surface.Changes in the index of refraction at the surface where the bindinginteraction occurs are detected by the hardware and recorded as RU(resonance units) in the control software. Curves are generated from theRU trace and are evaluated by fitting algorithms which compare the rawdata to well-defined binding models. These fits allow determination of avariety of thermodynamic constants, including the apparent affinity ofthe binding interaction. SPR main advantage is that it does not requirelabeling the protein or the binding compound.

The kinetics of enzymatic-catalyzed reactions is a useful tool not onlyto determine the inhibition constants (Ki) for an inhibitor but also thesite at which an inhibitor binds to the enzyme. An inhibitor that bindsexclusively to the catalytic active site displays a competitiveinhibition pattern with the substrate. In contrast, an inhibitor thatbinds to a different site from that of the substrate displays anuncompetitive inhibition pattern with the substrate. If the inhibitorbinds to both an active site and a different site from that of theactive site, it would display a non-competitive pattern. Thus, theactive ingredient (e.g. the compound of formula (I)) would becompetitive with the activation peptide and uncompetitive with thesubstrate.

NMR methods and optical spectroscopic methods such as fluorescence (e.g.fluorescence anisotropy (FA)), UV-vis, and circular dichroism (CD) arewell-known assays utilized in measuring the kinetics of the interactionbetween an active ingredient and a protein. In a preferred embodiment,fluorescence anisotropy is used.

The fluorescence method is suitable for high throughput screening methodamenable to automation in a laboratory environment. Since HCV NS3contains two tryptophan residues and four tyrosine residues, the bindingof an inhibitor (e.g. the compound of formula (I), NS4A) to theactivation site may be accompanied by significant change in theintrinsic fluorescence of the protein, and hence the enzyme inhibitionmay be evaluated (see FIG. 6). A peptide inhibitor (e.g. NS4A) may belabeled with a fluorescent probe and the binding of the labeled peptideto the enzyme is accompanied by fluorescent change (see Example 12, 13,FIG. 6). Another fluorescence assay method for determining the bindingconstant of the compound of formula (I) disclosed herein is acompetitive displacement assay (see Examples 11, 13, FIG. 5).

NMR methods may be used to observe the binding of an active ingredient(e.g. the compound of formula (I)) to the activation site of HCV NS3protease. In its simple form, the observation of broadening of an NMRsignal as a function of concentration would allow the determination ofbinding constants.

The examples below are intended to further illustrate protocols forpreparing, characterizing the compound of formulae (I) and (II), anduses thereof, and are not intended to limit the scope of the claims.

Example 1

Synthesis of Screened Compounds

The compounds were synthesized using straightforward chemistry [Duguay,G.; Guémas, J.-P.; Meslin, J.-C.; Pradere, J.-P.; Reliquet, F.;Reliquet, A.; Tea-Gokou, C.; Quiniou, H.; Rabiller, C., Heteroatomicchains and their products of cyclisation. IV.t-butyl-2-phthalimido-2-(3,6-dihydro-1,3-2H-thiazine-2-yliden)-acetatessubstituted in position 5 by a functional group. Journal of HeterocyclicChemistry 1980, 17 (4), 767-770; and Vuilhorgne, M.; Malpart, J.; Mutti,S.; Mignani, S., Preparative route to2-ethoxycarbonylimidazole-4-phosphonate anddiethylimidazole-2,4-dicarboxylate. Journal of Heterocyclic Chemistry2003, 40 (1), 159-162, each incorporated herein by reference in theirentirety] as illustrated in the synthesis scheme (FIG. 2). All finalcompounds are new and their structures were confirmed by spectralanalyses (e.g. ¹H NMR, ¹³C NMR, and LC/MS).

Example 2

Chemical Synthesis: General

Solvents and reagents were purchased from Sigma-Aldrich (USA), VWR (USA)or Alfa Aesar (UK). When needed, solvents were dried accordingprocedures described in literature. Unless stated otherwise, reactionswere performed under inert atmosphere of nitrogen. Melting points (m.p.)were determined in open capillary tubes using Electrothermal apparatus(Stuart, UK) and were uncorrected. NMR spectra were recorded on BrukerDPX-300 MHz (Bruker, Switzerland). HPLC-Mass Spectrometry was performedon Agilent 1100/ZQ MSD including diod-array UV detector.

Example 3 Synthesis of 5-(tert-butyl) 2-ethyl1H-imidazole-2,5-dicarboxylate (MOC-01)

Water (0.6 mL/mmol) was added dropwise over a period of 2 h to a stirredmixture of ethyl cyanoformate (1.0 equiv, 3 g, 30.3 mmol), hydroxylaminehydrochloride (1.5 equiv, 3.16 g, 45.45 mmol), and sodium carbonate(0.77 equiv, 2.47 g, 23.331 mmol) in ethanol (1 mL/mmol) at r.t. Uponcompletion, the reaction was quenched and the solvent was removed undervacuum. The resulting residue was extracted with DCM, and the combinedorganic layers were washed with brine, dried over MgSO₄, filtered, andconcentrated to afford a white solid. The intermediate(Z)-2-amino-2-(hydroxyimino) acetate was recrystallized from chloroformand n-heptane to afford white crystals (1.88 g, 47%). This intermediate(1.0 equiv, 132 mg, 1 mmol) was added to a solution of tBu-propiolate(1.0 equiv, 126 mg, 1 mmol), Et₃N (1.0 equiv, 101 mg, 1 mmol) andtoluene (5.5 mL/mmol). The resulting solution was MW irradiated at 120°C. for 3 min (300 W) and then for an additional 10 min. The solvent wasevaporated and the final product was purified via preparative HPLC. ¹HNMR (300 MHz, DMSO-d₆) d_(H) ppm 13.72 (br., 1H), 7.87 (s, 1H), 4.34 (q,J=7.18 Hz, 2H), 1.51 (s, 9H), 1.32 (t, J=6.99 Hz, 3H); ¹³C NMR (214 MHz,CDCl₃) d_(c) 169.7, 136.5, 129.5, 129.1, 128.9, 127.8, 123.8, 29.7; IR(FT-IR, cm⁻¹): 3025.37, 2916.82, 2843.61, 1695.04, 1619.31, 1594.07,1571.35, 1505.72, 1445.14, 1399.70, 1359.31, 1270.96, 1205.32; LC-MS(ESI), RT=1.41 min, m/z 241.3 [M+H]⁺.

Example 4 Synthesis of 2-(ethoxycarbonyl)-1H-imidazole-4-carboxylic acid(MOC-02)

A solution of 5-tert-butyl 2-ethyl 1H-imidazole-2,5-dicarboxylate (1equiv, 4.299 g, 17.6 mmol) and CF₃COOH (1.2 mL/mmol) in DCM (0.84mL/mmol) was stirred for 12 h at r.t. The solvent was evaporated undervacuum and the remaining residue was treated with MeOH (4 mL/mmol) andfiltered to give a yellow solid (2.841 g, 86.29%). ¹H NMR (300 MHz,DMSO-d₆) d_(H) ppm 13.72 (br. s, 1H); 12.59 (br. s, 1H), 7.91 (s, 1H),4.33 (q, J=6.97 Hz, 2H), 1.32 (t, J=7.06 Hz, 3H); LC-MS (ESI), RT=1.55min, m/z 185.3 [M+H]⁺.

Example 5 General Procedure for the Synthesis of ethyl4-(N-substitutedcarbamoyl)-1H-imidazole-2-carboxylate (MOC-03 to MOC-6)

Under inert atmosphere, the carboxylic acid MOC-02 (1.0 equiv., 5.21mmol) was dissolved in dry THF (5 mL/mmol) then EDCI (1.0 equiv, 0.998g, 5.21 mmol), HOBt (1.0 equiv, 0.704 mg, 5.21 mmol), DIPEA (1.5 equiv,1.01 g, 7.8 mmol), and the amine (1.2 equiv, 6.252 mmol) were addedorderly. The mixture was stirred at r.t. for 1 h then heated to 60° C.After 3 h, EDCI (0.5 equiv, 0.5 g, 2.6 mmol), HOBt (0.5 equiv, 0.352 g,2.6 mmol), DIPEA (0.75 equiv, 0.505 g, 3.9 mmol) were added to themixture. After completion, the mixture was purified by columnchromatography using 50-100% EtOAc in cyclohexane.

Ethyl4-((2-(methylamino)-2-oxoethyl)carbamoyl)-1H-imidazole-2-carboxylate(MOC-03)

Yield was 77%. ¹H NMR (300 MHz, DMSO-d₆) d_(H) ppm 8.19 (br. s., 1H),7.81 (s, 2H), 4.35 (q, J=7.16 Hz, 2H), 3.81 (d, J=5.65 Hz, 2H), 2.59 (d,J=4.52 Hz, 3H), 1.33 (t, J=7.16 Hz, 3H); LC-MS (ESI), RT=1.57 min, m/z255.3 [M+H]⁺.

Synthesis of (((4-aminobutyl)amino)(carboxyamino)methylene)carbamic acidditert-butyl ester (Amine Reactant for MOC-04)

To a solution of 1,4-diaminobutane (2.0 equiv, 303.24 mg, 3.44 mmol) inTHE (1.3 mL/mmol), a solution of1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (1.0 equiv, 500mg, 1.72 mmol) in THE (1.7 mL/mmol) was added within 0.5 h. The solutionwas stirred at room temperature for 1 h. After completion, the solventwas removed under vacuum and the product was purified by columnchromatography on silica gel using a mixture of DCM/MeOH to give(((4-aminobutyl)amino)(carboxyamino)methylene)carbamic acid ditert-butylester (250 mg, 52.88%); ¹H NMR (300 MHz, CDCl₃) d_(H) ppm 11.51 (br. s.,1H), 8.36 (br. s., 1H), 3.40-3.52 (m, 2H), 2.75 (t, J=6.69 Hz, 2H),1.51-1.71 (m, 4H), 1.52 (s, 9H), 1.51 (s, 9H); LC-MS (ESI), RT=2.44 min,m/z 331.8 [M+H]⁺.

Ethyl(E)-4-((4-(2,3-bis(tert-butoxycarbonyl)guanidino)butyl)carbamoyl)-1H-imidazole-2-carboxylate(MC-04)

Yield was 36%. The compound was used as the starting material for thenext step without further characterization.

Ethyl 5-((pyridin-2-ylmethyl)carbamoyl)-1H-imidazole-2-carboxylate(MOC-05)

Yield was 94%. ¹H NMR (300 MHz, DMSO-d₆) d_(H) ppm 13.24 (br. s. 1H),8.69 (t, J=5.65 Hz, 1H), 8.51 (d, J=4.14 Hz, 1H), 8.41 (s, 1H), 7.81 (s,1H), 7.74 (t, J=7.06 Hz, 1H), 7.19-7.35 (m, 2H), 4.53 (d, J=6.03 Hz,2H), 4.34 (q, J=7.16 Hz, 2H), 1.32 (t, J=7.06 Hz, 3H); LC-MS (ESI),RT=1.14 min, m/z 275.3 [M+H]⁺.

Ethyl 5-((furan-2-ylmethyl)carbamoyl)-1H-imidazole-2-carboxylate(MOC-06)

Yield was 76%. ¹H NMR (300 MHz, DMSO-d₆) d_(H) ppm 13.76 (br. s., 1H),8.53 (br. s., 1H), 7.82 (s, 1H), 7.50-7.58 (m, 1H), 6.22 (d, J=3.02 Hz,1H), 6.38 (dd, J=3.02, 1.89 Hz, 1H), 6.22 (d, J=3.02 Hz, 1H), 4.41 (d,J=6.04 Hz, 2H), 4.34 (q, J=7.18 Hz, 2H), 1.32 (t, J=6.99 Hz, 3H).

Example 6

General Procedure for the Synthesis of MOC-07 to MOC-10

The ethyl esters (1.0 equiv) was dissolved in TH/water 4:1 (4 mL/mmol)then LiOH (3.0 equiv) was added. The mixture was stirred at r.t. untilcompletion. Afterwards, the solvent was evaporated under vacuum. Theremaining residue was re-suspended in EtOH/toluene and evaporated. Nofurther purification or characterization was performed and the saltswere used for the next step.

Example 7

General Procedure for the Synthesis of MOC-11 to MOC-36

Under inert atmosphere, the appropriate lithium saltimidazole-carboxylic acid derivative of MOC-7 to MOC-10 (1.0 equiv.) wasdissolved in dry THE (3 mL/mmol) then propanephosphonic acid anhydride(T₃P, 50% solution in THE, 6.0 equiv.) and the requisite aminederivative (1.2 equiv.) were added. The mixture was stirred at r.t. for72 h. After 24 h, 6.0 equiv. of T₃P (50% solution in THF) was added tothe mixture. Upon reaction completion, the solvent was evaporated andthe residue was purified on a silica gel column with 0-20% MeOH inCDCl₃, and then the crude product was collected and purified by prepHPLC.

N⁵-(2-(methylamino)-2-oxoethyl)-N²-(n-pentyl)-1H-imidazole-2,5-dicarboxamide(MOC-11)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.76 (s, 1H), 4.04 (s, 1H), 3.74(t, J=6.42 Hz, 2H), 3.38-3.51 (m, 1H), 2.78 (s, 2H), 1.80-2.00 (m, 2H),1.53-1.80 (m, 2H), 1.20-1.53 (m, 3H), 0.86-1.07 (m, 3H); ¹³C NMR (75MHz, METHANOL-d₄) d_(C) ppm 170.8, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1,46.8, 41.8, 38.9, 29.0, 28.9, 25.0, 22.1, 13.0; LC-MS (ESI), RT=1.46min, m/z 296.4 [M+H]⁺.

N²-(n-hexyl)-N⁵-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-dicarboxamide(MOC-12)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.76 (s, 1H), 4.04 (s, 1H), 3.39(t, J=7.18 Hz, 1H), 2.78 (s, 2H), 1.56-1.91 (m, 3H), 1.38 (br. s., 5H),1.04 (t, J=6.42 Hz, 2H), 0.77-0.98 (m, 3H); ¹³C NMR (75 MHz,METHANOL-d₄) d_(C) ppm 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8,39.0, 31.3, 29.2, 26.3, 25.0, 22.3, 13.0; LC-MS (ESI), RT=1.39 min, m/z310.3 [M+H]⁺.

N²-(n-heptyl)-N⁵-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-dicarboxamide(MOC-13)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.68 (s, 1H), 3.95 (s, 2H), 2.69(s, 3H), 1.45-1.73 (m, 2H), 1.25 (s, 5H), 1.29 (s, 4H), 0.63-1.01 (m,4H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm 48.5, 48.2, 47.9, 47.6,47.4, 47.1, 46.8, 38.9, 31.6, 29.3, 28.8, 26.6, 25.0, 22.3, 13.0; LC-MS(ESI), RT=2.59 min, m/z 324.4 [M+H]⁺.

N²-(2-(cyclohexyloxy)ethyl)-N⁵-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-dicarboxamide(MOC-14)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.77 (s, 1H), 3.84-4.24 (m, 2H),3.66 (t, J=5.48 Hz, 2H), 3.56 (t, J=5.29 Hz, 2H), 2.77 (s, 3H), 1.93(br. s., 2H), 1.62-1.84 (m, 4H), 1.45-1.62 (m, 1H), 1.16-1.41 (m, 6H),0.98-1.12 (m, 1H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm 122.2, 77.7,65.8, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 39.3, 31.9, 25.5,25.0, 23.7; LC-MS (ESI), RT=1.60 min, m/z 352.3 [M+H]⁺.

N²-(3-(cyclohexyloxy)propyl)-N⁵-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-dicarboxamide(MOC-15)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.76 (s, 1H), 3.86-4.19 (m, 2H),3.41-3.70 (m, 4H), 2.78 (s, 3H), 1.83-2.03 (m, 4H), 1.44-1.83 (m, 5H),1.17-1.44 (m, 5H), 0.97-1.17 (m, 1H); ¹³C NMR (75 MHz, METHANOL-d₄)d_(C) ppm 77.6, 65.5, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.7,36.9, 31.9, 29.5, 25.6, 25.0, 23.7; LC-MS (ESI), RT=1.59 min, m/z 366.4[M+H]⁺.

N⁵-(2-(methylamino)-2-oxoethyl)-N²-(2-phenoxyethyl)-1H-imidazole-2,5-dicarboxamide(MOC-16)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.63-7.83 (m, 1H), 7.13-7.43 (m,2H), 6.79-7.13 (m, 3H), 4.11-4.30 (m, 2H), 4.03 (s, 2H), 3.80 (t, J=5.29Hz, 2H), 2.77 (s, 3H), 1.42 (t, J=6.99 Hz, 1H); ¹³C NMR (75 MHz,METHANOL-d₄) d_(C) ppm 129.1, 114.3, 48.5, 48.2, 47.9, 47.6, 47.3, 47.1,46.8, 24.9; LC-MS (ESI), RT=1.40 min, m/z 346.3 [M+H]⁺.

N⁵-(2-(methylamino)-2-oxoethyl)-N²-(3-phenoxypropyl)-1H-imidazole-2,5-dicarboxamide(MOC-17)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.65-7.88 (m, 1H), 7.16-7.37 (m,2H), 6.79-7.05 (m, 3H), 3.91-4.22 (m, 4H), 3.61 (t, J=6.80 Hz, 2H), 2.77(s, 3H), 2.11 (quin, J=6.33 Hz, 2H), 1.43 (t, J=6.99 Hz, 1H); ¹³C NMR(75 MHz, METHANOL-d₄) d_(C) ppm 129.1, 120.4, 114.2, 65.3, 48.5, 48.2,47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 36.4, 29.0, 25.0; LC-MS (ESI),RT=1.39 min, m/z 360.4 [M+H]⁺.

N²-(4-isopropoxybutyl)-N⁵-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-dicarboxamide(MOC-18)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.76 (s, 1H), 3.91-4.25 (m, 2H),3.56-3.78 (m, 1H), 3.35-3.56 (m, 4H), 2.78 (s, 3H), 1.55-1.87 (m, 8H),1.11-1.26 (m, 7H), 1.05 (t, J=6.61 Hz, 3H); ¹³C NMR (75 MHz,METHANOL-d₄) d_(C) ppm 67.4, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8,38.7, 27.1, 26.1, 25.0, 21.0; LC-MS (ESI), RT=1.79 min, m/z 340.4[M+H]⁺.

N⁵-(2-(methylamino)-2-oxoethyl)-N²-(6-methylheptyl)-1H-imidazole-2,5-dicarboxamide(MOC-19)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.77 (s, 1H), 4.04 (s, 2H), 2.78(s, 3H), 1.63 (br. s., 1H), 1.25-1.55 (m, 9H), 0.76-1.11 (m, 7H); ¹³CNMR (75 MHz, METHANOL-d₄) δ ppm 48.5, 48.2, 47.9, 47.6, 47.4, 47.1,46.8, 42.1, 39.5, 30.6, 28.6, 25.0, 23.8, 22.7, 13.0, 9.8; LC-MS (ESI),RT=2.66 min, m/z 338.4 [M+H]⁺.

(E)-N²-(3,7-dimethylocta-2,6-dien-1-yl)-N⁵-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-dicarboxamide(MOC-20)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.76 (s, 1H), 5.31 (t, J=6.42Hz, 1H), 5.12 (t, J=6.23 Hz, 1H), 3.86-4.17 (m, 4H), 2.78 (s, 3H),1.94-2.24 (m, 4H), 1.76 (s, 3H), 1.62 (s, 3H), 1.67 (s, 3H); ¹³C NMR (75MHz, METHANOL-d₄) d_(C) ppm 123.7, 119.9, 48.5, 48.2, 47.9, 47.6, 47.4,47.1, 46.8, 41.8, 39.3, 36.7, 26.1, 25.0, 24.5, 16.4, 15.0; LC-MS (ESI),RT=1.60 min, m/z 362.4 [M+H]⁺.

N⁵-(2-(methylamino)-2-oxoethyl)-N²-(3-methylpentyl)-1H-imidazole-2,5-dicarboxamide(MOC-21)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.76 (s, 1H), 4.04 (s, 2H),3.38-3.56 (m, 2H), 2.78 (s, 3H), 1.59-1.84 (m, 2H), 1.34-1.59 (m, 2H),1.16-1.34 (m, 1H), 0.76-1.16 (m, 7H); ¹³C NMR (75 MHz, METHANOL-d₄)d_(C) ppm 170.8, 122.1, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8,37.1, 35.9, 32.1, 29.1, 25.0, 18.0, 10.2; LC-MS (ESI), RT=1.39 min, m/z310.4 [M+H]⁺.

N²-(2-acetamidoethyl)-N⁵-(2-(methylamino)-2-oxoethyl)-1H-imidazole-2,5-dicarboxamide(MOC-22)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.76 (s, 1H), 4.04 (s, 1H),3.35-3.65 (m, 3H), 2.78 (s, 2H), 1.88-2.07 (m, 3H), 1.66 (d, J=8.67 Hz,2H), 1.04 (t, J=6.03 Hz, 1H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 41.8, 39.1, 38.7, 25.0, 21.2;LC-MS (ESI), RT=2.82 min, m/z 311.33 [M+H]⁺.

N⁵-(4-guanidinobutyl)-N²-(n-pentyl)-1H-imidazole-2,5-dicarboxamide(MOC-23)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.83 (s, 1H), 3.36-3.60 (m, 5H),3.25 (br. s., 3H), 1.69 (br. s., 7H), 1.17-1.50 (m, 6H), 0.95 (br. s.,4H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm 48.5, 48.3, 48.0, 47.7,47.4, 47.1, 46.8, 40.8, 39.3, 28.9, 28.7, 25.9, 22.1, 13.0; LC-MS (ESI),RT=1.90 min, m z 338.4 [M+H]⁺.

N⁵-(4-guanidinobutyl)-N²-(n-hexyl)-1H-imidazole-2,5-dicarboxamide(MOC-24)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.81 (br. s., 1H), 3.39-3.57 (m,4H), 3.07-3.27 (m, 3H), 1.61-1.84 (m, 8H), 1.55 (t, J=7.16 Hz, 1H),1.18-1.48 (m, 9H), 0.93 (br. s., 4H); ¹³C NMR (75 MHz, METHANOL-d₄)d_(C) ppm 48.5, 48.2, 48.0, 47.7, 47.4, 47.1, 46.8, 40.8, 39.2, 31.3,26.4, 25.9, 22.3, 13.0; LC-MS (ESI), RT=1.78 min, m/z 352.3 [M+H]⁺.

N⁵-(4-guanidinobutyl)-N²-(n-heptyl)-1H-imidazole-2,5-dicarboxamide(MOC-25)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.90 (s, 1H), 3.35-3.57 (m, 5H),3.25 (br. s., 2H), 1.60-1.86 (m, 7H), 1.34 (s, 6H), 1.39 (s, 4H),0.79-1.03 (m, 4H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm 48.6, 48.3,48.0, 47.7, 47.4, 47.1, 46.9, 40.8, 39.6, 38.5, 31.6, 28.9, 28.7, 26.7,26.3, 25.9, 22.3, 13.1; LC-MS (ESI), RT=1.78 min, m/z 366.4 [M+H]⁺.

N²-(2-(cyclohexyloxy)ethyl)-N⁵-(4-guanidinobutyl)-1H-imidazole-2,5-dicarboxamide(MOC-26)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.76 (br. s., 1H), 3.50-3.75 (m,7H), 3.44 (br. s., 3H), 3.25 (br. s., 3H), 1.81-2.09 (m, 5H), 1.63-1.81(m, 9H), 1.45-1.63 (m, 2H), 1.11-1.44 (m, 7H); ¹³C NMR (75 MHz,METHANOL-d₄) d_(C) ppm 77.5, 65.6, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1,46.8, 40.7, 37.0, 31.9, 25.8, 25.6, 23.7; LC-MS (ESI), RT=2.46 min, m/z213.2 [M+H]⁺.

N²-(3-(cyclohexyloxy)propyl)-N⁵-(4-guanidinobutyl)-1H-imidazole-2,5-dicarboxamide(MOC-27)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.73 (s, 1H), 3.38-3.76 (m, 7H),3.13-3.30 (m, 3H), 1.79-2.07 (m, 5H), 1.63-1.79 (m, 7H), 1.56 (d, J=7.54Hz, 1H), 1.12-1.42 (m, 5H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm77.5, 65.6, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.7, 37.0, 31.9,25.8, 25.6, 23.7; LC-MS (ESI), RT=1.98 min, m/z 408.3 [M+H]⁺.

N⁵-(4-guanidinobutyl)-N²-(2-phenoxyethyl)-1H-imidazole-2,5-dicarboxamide(MOC-28)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 8.01 (s, 1H), 7.28 (t, J=7.91Hz, 2H), 6.82-7.10 (m, 3H), 4.19 (t, J=5.09 Hz, 2H), 3.84 (t, J=5.09 Hz,2H), 3.44 (br. s., 2H), 3.25 (br. s., 2H), 1.69 (br. s., 5H); ¹³C NMR(75 MHz, METHANOL-d₄) d_(C) ppm 129.2, 114.2, 48.5, 48.2, 47.9, 47.6,47.4, 47.1, 46.8; LC-MS (ESI), RT=1.86 min, m/z 388.4 [M+H]⁺.

N⁵-(4-guanidinobutyl)-N²-(3-phenoxypropyl)-1H-imidazole-2,5-dicarboxamide(MOC-29)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.73 (s, 1H), 7.13-7.38 (m, 2H),6.80-7.05 (m, 3H), 4.10 (t, J=5.93 Hz, 2H), 3.61 (t, J=6.78 Hz, 2H),3.44 (br. s., 2H), 3.14-3.30 (m, 2H), 2.11 (quin, J=6.36 Hz, 2H),1.52-1.84 (m, 5H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm 129.1, 120.4,114.2, 65.3, 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.8, 37.9, 36.4,29.0, 26.6, 25.8; LC-MS (ESI), RT=1.51 min, m/z 402.3 [M+H]⁺.

N⁵-(4-guanidinobutyl)-N²-(4-isopropoxybutyl)-1H-imidazole-2,5-dicarboxamide(MOC-30)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.73 (s, 1H), 3.35-3.74 (m, 8H),3.14-3.28 (m, 2H), 1.69 (br. s., 9H), 1.16 (d, J=6.22 Hz, 6H); ¹³C NMR(75 MHz, METHANOL-d₄) d_(C) ppm 71.5, 67.4, 48.5, 48.2, 47.9, 47.6,47.4, 47.1, 46.8, 40.8, 38.7, 37.9, 27.1, 26.6, 26.1, 25.8, 21.0; LC-MS(ESI), RT=2.08 min, m/z 382.3 [M+H]⁺.

N⁵-(4-guanidinobutyl)-N²-(6-methylheptyl)-1H-imidazole-2,5-dicarboxamide(MOC-31)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.74 (s, 1H), 3.38-3.57 (m, 2H),3.14-3.30 (m, 2H), 1.54-1.83 (m, 6H), 1.22-1.54 (m, 9H), 0.81-1.09 (m,7H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm 48.5, 48.2, 47.9, 47.6,47.3, 47.1, 46.8, 40.8, 39.5, 30.6, 28.6, 26.7, 25.8, 23.8, 22.7, 13.0,9.8; LC-MS (ESI), RT=1.19 min, m z 380.3 [M+H]⁺.

N⁵-(4-guanidinobutyl)-N²-(3-methylpentyl)-1H-imidazole-2,5-dicarboxamide(MOC-32)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.73 (s, 1H), 3.35-3.58 (m, 4H),3.14-3.30 (m, 2H), 1.69 (br. s., 6H), 1.35-1.59 (m, 3H), 1.24 (td,J=7.06, 13.75 Hz, 1H), 0.77-1.09 (m, 6H); ¹³C NMR (75 MHz, METHANOL-d₄)d_(C) ppm 48.5, 48.2, 47.9, 47.6, 47.4, 47.1, 46.8, 40.8, 37.1, 35.9,32.1, 29.1, 26.6, 25.8, 18.0, 10.2; LC-MS (ESI), RT=1.79 min, m/z 352.3[M+H]⁺.

N²-(n-hexyl)-N⁵-(pyridin-2-ylmethyl)-1H-imidazole-2,5-dicarboxamide(MOC-33)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 8.52 (d, J=4.53 Hz, 1H),7.70-7.93 (m, 2H), 7.45 (d, J=7.93 Hz, 1H), 7.19-7.40 (m, 1H), 4.71 (s,2H), 3.39 (t, J=6.99 Hz, 2H), 1.51-1.76 (m, 2H), 1.37 (br. s., 6H), 0.93(t, J=6.42 Hz, 3H); ¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm 158.6,157.8, 148.5, 137.5, 122.5, 121.9, 121.6, 48.5, 48.2, 47.9, 47.7, 47.4,47.1, 46.8, 43.7, 39.0, 31.3, 29.2, 26.3, 22.3; LC-MS (ESI), RT=2.74min, m/z 330.3 [M+H]⁺.

N²-(2-acetamidoethyl)-N⁵-(pyridin-2-ylmethyl)-1H-imidazole-2,5-dicarboxamide(MOC-34)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 8.65 (d, J=4.91 Hz, 1H),8.08-8.32 (m, 1H), 7.69-7.93 (m, 2H), 7.54-7.69 (m, 1H), 3.47 (dd,J=5.85, 19.83 Hz, 4H), 1.97 (s, 3H), 0.92-1.19 (m, 1H); ¹³C NMR (75 MHz,METHANOL-d₄) d_(C) ppm 142.1, 124.0, 123.6, 48.5, 48.2, 47.9, 47.7,47.4, 47.1, 46.8, 39.3, 38.7, 21.2; LC-MS (ESI), RT=3.18 min, m/z 331.3[M+H]⁺.

N⁵-(furan-2-ylmethyl)-N²-(n-hexyl)-1H-imidazole-2,5-dicarboxamide(MOC-35)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.75 (s, 1H), 7.45 (s, 1H),6.18-6.55 (m, 2H), 4.57 (s, 2H), 3.36-3.42 (m, 2H), 1.61 (d, J=7.18 Hz,2H), 1.37 (br. s., 7H), 0.76-1.06 (m, 3H); ¹³C NMR (75 MHz, METHANOL-d₄)d_(C) ppm 158.6, 151.7, 142.0, 121.8, 110.0, 106.8, 48.5, 48.2, 47.9,47.7, 47.4, 47.1, 46.8, 38.9, 35.4, 31.3, 29.2, 26.3, 22.3, 13.0; LC-MS(ESI), RT=2.43 min, m/z 319.3 [M+H]⁺.

N²-(2-acetamidoethyl)-N⁵-(furan-2-ylmethyl)-1H-imidazole-2,5-dicarboxamide(MOC-36)

¹H NMR (300 MHz, METHANOL-d₄) d_(H) ppm 7.75 (s, 1H), 7.45 (s, 1H), 6.32(s, 1H), 6.37 (s, 1H), 4.57 (s, 2H), 3.35-3.65 (m, 5H), 1.96 (s, 3H);¹³C NMR (75 MHz, METHANOL-d₄) d_(C) ppm 172.6, 159.0, 151.8, 142.0,110.0, 106.8, 48.5, 48.2, 47.9, 47.7, 47.4, 47.1, 46.8, 39.0, 38.7,35.4, 21.2; LC-MS (ESI), RT=1.71 min, m/z 319.13 [M+H]⁺.

Example 8

Biological Screening: General

All reagents used in the biological screenings were purchased fromSigma-Aldrich (UK) of molecular biology grade unless stated otherwise.

Example 9

NS3 Protein

(i) NS3 Constructs

A synthetic gene coding for the HCV NS3 domain of genotype 4a, the mostabundant HCV in Saudi Arabia and Egypt [Massariol, M.-J.; Zhao, S.;Marquis, M.; Thibeault, D.; White, P. W., Protease and helicaseactivities of hepatitis C virus genotype 4, 5, and 6 NS3-NS4A proteins.Biochemical and Biophysical Research Communications 2010, 391 (1),692-697], was synthesized by GenScript (Hong Kong), the nucleotidesequence was optimized for E. coli codon usage. The synthetic gene wascloned as NdeI-BamHI fragment into the expression vector pET-3aNovagen®. The obtained construct was sequenced to confirm that we hadthe right clone and the gene was in the correct frame.

(ii) NS3 Protein Information

Accession GU085486.1

HCV genotype 4a (The most common genotype in Saudi Arabia) [Bawazir, A.;AlGusheri, F.; Jradi, H.; AlBalwi, M.; Abdel-Gader, A.-G., Hepatitis Cvirus genotypes in Saudi Arabia: a future prediction and laboratoryprofile. Virology journal 2017, 14 (1), 208-208]NS3 from 4 to 182 aa (L/E, F/E, I/Q, V/E, L/Q, C/S)NS4A 632 to 685 aa (i/n)G svvivgrvnl sgdtayaqqt rgeestqets qtgrdtnenc gevqvlstat qsflgtavngvmwtvyhgag sktisgpkgp vnqmytnvdq dlvgwpsppg vksltpctcg asdlylvtrhadvvpvrrrg dtrgallspr pistlkgssg gpllcpmgha aglfraavst rgvakavdfvpveslett mrspNS4A/NS3 Fusion protein expression in pET-28aNS3 protease domain 1-181 aa+N-terminal T7 tag and C-terminal His tagM ASMTGGQQMG apitayaqqt rglfstivts ltgrdtnenc gevqvlstat qsflgtavngvmwtvyhgag sktisgpkgp vnqmytnvdq dlvgwpsppg vksltpctcg asdlylvtrhadvvpvrrrg dtrgallspr pistlkgssg gpllcpmgha aglfraavct rgvakavdfvpveslettmr sGSHHHHHHExpression in pET-3a(iii) Protein eNS4A Expression

The sequence of NS3 domain for genotype 4a, was expressed in E. coliRosette (DE3) pLysS according to standard protocol [Kim, J.;Morgenstern, K.; Lin, C.; Fox, T.; Dwyer, M.; Landro, J.; Chambers, S.;Markland, W.; Lepre, C.; O'malley, E., Crystal structure of thehepatitis C virus NS3 protease domain complexed with a synthetic NS4Acofactor peptide. Cell 1996, 87 (2), 343-355]. Therefore, a syntheticgene for NS3 domain was subcloned in the expression vector pET-3a. Inthe process, a 100 mL of bacterial culture in Luria Broth medium wasgrown overnight at 37° C. and used for inoculation of 10 L LB in a14-liter fermenter flask (New Brunswick Scientific Co., CT, USA). Themedia was supplemented with 50 μg/mL ampicillin. The culture grew untilthe OD₆₀₀ reached 0.5-0.6, then it was cooled to 25° C. and 1 mM IPTGwas added. Expression was followed overnight, and then cells wereharvested.

(iv) Protein Purification

The produced protein was purified using equilibrated Ni-NTA beads andthe poly-histidine tag was not removed. In the process, cells werere-suspended (1 g/5 mL) in buffer (50 mM HEPES, 0.3 M NaCl, 10%glycerol, 2 mM β-mercaptoethanol, pH 8). Lysozymes were added (1 mg/mL)followed by protease inhibitor cocktail tablet and the suspension wassonicated. Cell lysate was centrifuged to collect the clear supernatantthat contained the desired NS3 protein. The protein was purified usingpre-equilibrated Ni-NTA beads (Qiagen, USA). Beads were washed withbuffer (50 mM HEPES, 0.3 M NaCl, 10% glycerol, 2 mM β-mercaptoethanol,20 mM imidazole, pH 8) and eluted with another buffer (50 mM HEPES, 0.3M NaCl, 10% glycerol, 2 mM β-mercaptoethanol, 350 mM imidazole, pH 8).Fractions were collected and concentrated using Amicon Ultra-4 3000 MWCOcentrifugal device (Millipore, Germany). Protein purity afterNi-affinity purification step was not less than 70%. The purity, asestimated by SDS-PAGE, was sufficient to perform all investigations ofthis study and the protein was stable for several hours at testconditions (see FIG. 4) [Massariol, M.-J.; Zhao, S.; Marquis, M.;Thibeault, D.; White, P. W., Protease and helicase activities ofhepatitis C virus genotype 4, 5, and 6 NS3-NS4A proteins. Biochemicaland Biophysical Research Communications 2010, 391 (1), 692-697]. Theconcentration of NS3 in the final concentrate was measured usingNanodrop™ nanoscale spectrophotometer.

When needed, further purification of the protein was accomplished onSuperdex 75 16/90 column (GE Healthcare, USA) equilibrated in 20 mMHEPES, 10 mM DDT, 200 mM NaCl, pH 7.6 run at rate of 1 mL/min followedby SDS-PAGE for purity estimation.

Example 10

NS4A

The cofactor NS4A and the fluorescent fluorescein isothiocyanate NS4A(FITC-NS4A) were purchased from GenScript (Hong Kong). NS4A structurewas identical to that of HCV genotype 4a with two lysine residues addedat both the N- and C-termini. Thus the structure of NS4A used in thisstudy was LL-G₂₁SVVIVGRIVLSG₃₃-LL.

Example 11

Binding and Competition Assay

Binding buffer (20 mM HEPES, 10 mM DTT, 200 mM NaCl, pH 7.6), a mixtureof NS3 (1.8 μM) and FITC-NS4A (0.1 M) at the calculated affinityconstant concentration were placed in a 96-well plate as binding testsolution. A serial dilution of MOC compounds (dissolved in 1.5% DMSO inbinding buffer) (1/2 dilution starting from 150 μM to 0.292 μM) wasmixed gently with the binding text solution for 60 minutes in the dark.The dissociation constant was calculated according to the recommendedequation embedded in GraphPad Prism v8.

Example 12

Enzyme Inhibition Assay

The assay was performed using SensoLyte-520® HCV protease assay kitFluorometric® (Anaspec, Fremont, Calif., USA) according to a modifiedprocedure to suit the purpose of determination of allosteric inhibition.NS3 (4.0 μM) was mixed with MOC-23 (6.3 μM) for 15 minutes. Afterwards,5-FAM/QXL™520 fluorescence resonance energy transfer (FRET) peptide wasadded as instructed by the assay kit manual. The sequence of this FRETpeptide (5-FAM-SLGRKIQIQ) is derived from the cleavage site ofNS4A/NS4B. In the FRET peptide, the fluorescence of 5-FAM is quenched byQXL™520. Upon cleavage into two separate fragments by HCV NS3/4Aprotease, the fluorescence of 5-FAM is recovered, and can be monitoredat 490 nm/520 nm (excitation/emission). Controls included buffer,compound, compound+NS3, NS3+NS4A and FRET peptide separately. All testwells and controls were repeated in triplicates at the same 96-wellplate.

Example 13

Biological Screening Results

The binding affinity test was performed using fluorescence anisotropytechnique. In this test, maximum fluorescence was first determined atoptimal ratio of NS3 (1.8 μM) and FITC-NS4A (0.1 μM). Compounds thatcompete with NS4A should be able to decrease the fluorescence of thismixture because they prevent the binding to occur and it should beconcentration-dependent. Results showed some compounds suppressed thefluorescence emitted upon binding of NS4A with NS3. However, we foundthat MOC-23 showed highest affinity and more reproducible results thanother compounds (see FIG. 5). It could effectively compete withFITC-NS4A at dissociation constant K_(d)=6.303±0.3 μM). Thereproducibility was checked using another batch of the express NS3protein following the same procedure and the value wascalculated=7.815±1.014 μM. Compound MOC-24 showed binding affinity atsimilar level but results were less reproducible and standard error washigher. Therefore, compound MOC-23 was tested for its ability to inhibitthe NS3 enzyme. MOC-23 at its binding affinity concentration (6.3 μM)was able to inhibit NS3 and abolished any proteolytic activity of theenzyme (see FIG. 6).

The invention claimed is:
 1. A composition, comprising: non-structuralprotein 3 (NS3), and a compound of formula (I)

a salt thereof, a solvate thereof, or a mixture thereof, wherein: R₁ isn-pentyl; and m is 4, wherein the compound of formula (I) is bonded toan active site of the NS3 to inhibit protease activity of the NS3. 2.The composition of claim 1, further comprising: a pharmaceuticallyacceptable carrier and/or excipient.
 3. The composition of claim 2,wherein the pharmaceutically acceptable carrier and/or excipient is atleast one selected from the group consisting of a buffer, an inorganicsalt, a fatty acid, a vegetable oil, a synthetic fatty ester, asurfactant, and a polymer.
 4. The composition of claim 2, furthercomprising an antiviral agent.
 5. The composition of claim 2, furthercomprising a body fluid.